1 00:00:16,180 --> 00:00:18,430 BARBARA IMPERIALI: So we're going to get started. 2 00:00:18,430 --> 00:00:20,740 This is a complicated lecture to choreograph, 3 00:00:20,740 --> 00:00:24,190 but I'm going to do my very, very best because I think 4 00:00:24,190 --> 00:00:26,880 there's some pretty amazing stuff 5 00:00:26,880 --> 00:00:30,560 that we have to explain that is carried out in nature. 6 00:00:30,560 --> 00:00:34,600 And one of those things is how do we replicate the entire 7 00:00:34,600 --> 00:00:40,060 genome of organisms in one fell swoop almost perfectly-- 8 00:00:40,060 --> 00:00:42,070 sometimes there are little errors-- 9 00:00:42,070 --> 00:00:43,570 and fast. 10 00:00:43,570 --> 00:00:46,030 So we're going to refer to these numbers in a moment. 11 00:00:46,030 --> 00:00:48,040 But first of all, I just want to get you back 12 00:00:48,040 --> 00:00:51,970 in the picture of where we were at the end of the last section, 13 00:00:51,970 --> 00:00:53,680 the biochemistry section. 14 00:00:53,680 --> 00:00:55,630 And in the next few classes we're 15 00:00:55,630 --> 00:01:00,290 going to address issues related to the central dogma 16 00:01:00,290 --> 00:01:03,970 and the use of nucleic acids for information storage 17 00:01:03,970 --> 00:01:05,830 and information transfer. 18 00:01:05,830 --> 00:01:07,360 And before I do that, I just want 19 00:01:07,360 --> 00:01:10,630 to highlight a couple of things, just some terms 20 00:01:10,630 --> 00:01:15,190 that you should recall and recap from the biochemistry section 21 00:01:15,190 --> 00:01:17,210 about nucleic acids. 22 00:01:17,210 --> 00:01:21,520 So nucleic acids form complementary double strands 23 00:01:21,520 --> 00:01:24,340 through base pairing, and that base pairing 24 00:01:24,340 --> 00:01:28,360 involves hydrogen bonding between the nucleobases, 25 00:01:28,360 --> 00:01:32,860 one purine to one pyrimidine, and the AT base pair 26 00:01:32,860 --> 00:01:35,210 is worth two hydrogen bonds. 27 00:01:35,210 --> 00:01:38,590 The GC base pair is worth three hydrogen bonds. 28 00:01:38,590 --> 00:01:41,350 And those are actually pretty useful facts 29 00:01:41,350 --> 00:01:44,410 to remember because they tell us about the stability 30 00:01:44,410 --> 00:01:48,760 of double-stranded DNA, where it's easier to tease it apart, 31 00:01:48,760 --> 00:01:50,360 and other characteristics. 32 00:01:50,360 --> 00:01:53,270 So it's kind of a useful thing to keep in mind. 33 00:01:53,270 --> 00:01:56,110 So the base pairing between what-- 34 00:01:56,110 --> 00:02:00,370 the purine pyrimidine pair, which sets the exact register, 35 00:02:00,370 --> 00:02:02,070 down the double-stranded DNA. 36 00:02:02,070 --> 00:02:05,410 The backbone is the exact distance apart 37 00:02:05,410 --> 00:02:09,220 because you always combine a small pyrimidine 38 00:02:09,220 --> 00:02:11,570 with a much larger purine. 39 00:02:11,570 --> 00:02:15,130 So that's something you should feel comfortable about. 40 00:02:15,130 --> 00:02:19,990 The components are organized in an anti-parallel orientation, 41 00:02:19,990 --> 00:02:23,750 so one end runs 5 prime to 3 prime. 42 00:02:23,750 --> 00:02:26,310 The other end runs 3 prime to 5 prime. 43 00:02:26,310 --> 00:02:29,020 And I showed you last time an image 44 00:02:29,020 --> 00:02:32,740 that allows us to state quite clearly 45 00:02:32,740 --> 00:02:36,460 that the anti-parallel orientation is significantly 46 00:02:36,460 --> 00:02:39,640 more stable than the parallel orientation 47 00:02:39,640 --> 00:02:42,670 because you can't make all those great hydrogen bonds well 48 00:02:42,670 --> 00:02:44,860 in the parallel organization apart 49 00:02:44,860 --> 00:02:48,340 from everything else that becomes complicated. 50 00:02:48,340 --> 00:02:50,260 Another important thing that you're 51 00:02:50,260 --> 00:02:53,770 going to have to remember, especially in this lecture, 52 00:02:53,770 --> 00:02:57,370 is that we add new nucleotides to the 3 53 00:02:57,370 --> 00:03:00,370 prime end of a nucleic acid. 54 00:03:00,370 --> 00:03:03,550 And so I'm just going to put this little guy in the corner 55 00:03:03,550 --> 00:03:04,150 here. 56 00:03:04,150 --> 00:03:07,670 This is the ribose sugar. 57 00:03:07,670 --> 00:03:10,055 That would be where the nucleobase is attached. 58 00:03:12,920 --> 00:03:15,740 And it's a deoxy sugar, so there's 59 00:03:15,740 --> 00:03:19,050 no substituent at that carbon. 60 00:03:19,050 --> 00:03:22,880 This is the one position where the base is. the 2 position, 61 00:03:22,880 --> 00:03:27,260 where it's deoxy, the 3 position. 62 00:03:27,260 --> 00:03:28,820 And these are all prime numbers. 63 00:03:28,820 --> 00:03:31,430 Remember, the sugars have prime numbers. 64 00:03:31,430 --> 00:03:34,070 The bases themselves have numbers without primes 65 00:03:34,070 --> 00:03:36,650 to distinguish what we're talking about. 66 00:03:36,650 --> 00:03:42,970 So 3, 4, 5, and they're all prime. 67 00:03:42,970 --> 00:03:47,350 And when we grow the single strand of DNA, 68 00:03:47,350 --> 00:03:51,970 we always add new basis to the 3 prime end. 69 00:03:51,970 --> 00:03:53,670 That's an important numbering system. 70 00:03:56,310 --> 00:03:58,890 I don't like that piece of chalk. 71 00:03:58,890 --> 00:04:04,335 So we always grow from 5 prime to 3 prime. 72 00:04:09,340 --> 00:04:11,030 And that will be important to you 73 00:04:11,030 --> 00:04:13,520 as we go through the discussion today. 74 00:04:13,520 --> 00:04:18,110 And then finally, a feature of double-stranded DNA, 75 00:04:18,110 --> 00:04:21,649 quite unlike proteins, which sometimes you'll 76 00:04:21,649 --> 00:04:26,120 thermally or with pH melt out and will often 77 00:04:26,120 --> 00:04:29,990 have a problem reforming their reliable structure because 78 00:04:29,990 --> 00:04:34,010 instead of the structure refolding, they aggregate. 79 00:04:34,010 --> 00:04:36,200 Double-stranded DNA doesn't aggregate 80 00:04:36,200 --> 00:04:38,750 because it's got all the negative charges that 81 00:04:38,750 --> 00:04:40,550 would be quite repulsive. 82 00:04:40,550 --> 00:04:43,550 So it would be very difficult for DNA strands 83 00:04:43,550 --> 00:04:45,140 to aggregate as such because there's 84 00:04:45,140 --> 00:04:48,140 too much of a concentration of negative charges. 85 00:04:48,140 --> 00:04:51,710 That would be repulsion amongst the same type charges. 86 00:04:51,710 --> 00:04:55,200 So DNA can be peeled apart with heat, 87 00:04:55,200 --> 00:04:57,980 and it will reanneal faithfully to its partner 88 00:04:57,980 --> 00:04:59,720 a complementary strand. 89 00:04:59,720 --> 00:05:02,840 Once you get to six, seven, eight base pairs, 90 00:05:02,840 --> 00:05:05,960 that is a nice interaction between the strands, 91 00:05:05,960 --> 00:05:07,640 and it forms faithfully. 92 00:05:07,640 --> 00:05:10,100 If we have a mistake in the strands, 93 00:05:10,100 --> 00:05:13,420 the stability of the double strand will be a bit less, 94 00:05:13,420 --> 00:05:16,100 and we can measure that through physical measurements 95 00:05:16,100 --> 00:05:17,720 that we carry out in the lab. 96 00:05:17,720 --> 00:05:20,850 So we can measure the denaturation 97 00:05:20,850 --> 00:05:23,210 or the thermal melt temperature. 98 00:05:23,210 --> 00:05:29,690 So we call this process, after you 99 00:05:29,690 --> 00:05:33,470 separate double-stranded DNA, we call it reannealing, 100 00:05:33,470 --> 00:05:36,290 or it's also designated as hybridization 101 00:05:36,290 --> 00:05:39,290 to form a hybrid, which is the composite of the two 102 00:05:39,290 --> 00:05:40,400 structures. 103 00:05:40,400 --> 00:05:43,010 Those terms are used fairly interchangeably. 104 00:05:43,010 --> 00:05:43,510 All right. 105 00:05:43,510 --> 00:05:45,730 So the goals in the next four classes 106 00:05:45,730 --> 00:05:49,700 are to show you how the structures of the nucleic acids 107 00:05:49,700 --> 00:05:52,940 really are purposed for the sorts of processes 108 00:05:52,940 --> 00:05:54,260 that they undergo. 109 00:05:54,260 --> 00:05:57,680 So the replication of DNA, the conversion 110 00:05:57,680 --> 00:06:01,610 of a strand of DNA into complementary messenger 111 00:06:01,610 --> 00:06:05,270 RNA once the process of protein synthesis 112 00:06:05,270 --> 00:06:08,550 needs to be initiated, that second step. 113 00:06:08,550 --> 00:06:11,510 The first step is called replication. 114 00:06:11,510 --> 00:06:16,340 The second step, when you transcribe DNA into RNA, 115 00:06:16,340 --> 00:06:18,110 is called transcription. 116 00:06:18,110 --> 00:06:22,310 And then finally, when you translate messenger RNA 117 00:06:22,310 --> 00:06:23,280 into proteins. 118 00:06:23,280 --> 00:06:25,550 So going to a completely new language, 119 00:06:25,550 --> 00:06:27,200 we call it a translation. 120 00:06:27,200 --> 00:06:30,500 And that's the basis of these lectures. 121 00:06:30,500 --> 00:06:32,870 There is a little bit of stuff in these lectures 122 00:06:32,870 --> 00:06:36,710 about more complicated issues in the mammalian cell, where 123 00:06:36,710 --> 00:06:39,860 we have to process the messenger RNA a bit 124 00:06:39,860 --> 00:06:42,200 before we can have it leave the nucleus. 125 00:06:42,200 --> 00:06:45,680 And I'll explain at the time when it comes. 126 00:06:45,680 --> 00:06:48,500 So this is the lineup for the four lectures. 127 00:06:48,500 --> 00:06:50,680 All right. 128 00:06:50,680 --> 00:06:53,490 Well, I already talked to you how when you add-- 129 00:06:53,490 --> 00:06:55,110 I mentioned it over here-- 130 00:06:55,110 --> 00:07:01,520 when you add cytidine triphosphate, GTP, ATP, TTP, 131 00:07:01,520 --> 00:07:04,910 these are the activated nucleobases, 132 00:07:04,910 --> 00:07:10,520 so they're the deoxy nucleoside triphosphates. 133 00:07:10,520 --> 00:07:14,180 They are nucleotides because they include phosphate, 134 00:07:14,180 --> 00:07:16,970 but when we describe them, we'll call 135 00:07:16,970 --> 00:07:19,960 them nucleoside triphosphates if we're going 136 00:07:19,960 --> 00:07:22,640 to mention the phosphate part. 137 00:07:22,640 --> 00:07:25,640 I know that probably doesn't make any sense at all. 138 00:07:25,640 --> 00:07:27,780 Is everyone OK with that? 139 00:07:27,780 --> 00:07:31,170 If you're just generically describing something 140 00:07:31,170 --> 00:07:34,050 with a phosphate in it, you call it a nucleotide. 141 00:07:34,050 --> 00:07:36,270 If you want to be a little bit more specific, 142 00:07:36,270 --> 00:07:39,870 you say nucleoside, and then you say how many phosphates. 143 00:07:39,870 --> 00:07:40,530 All right? 144 00:07:40,530 --> 00:07:44,310 And so for the building blocks, it's a nucleoside triphosphate. 145 00:07:44,310 --> 00:07:46,710 And believe me, I get that wrong all the time, 146 00:07:46,710 --> 00:07:48,510 and my students correct me. 147 00:07:48,510 --> 00:07:51,330 So I don't have a problem if you're not 148 00:07:51,330 --> 00:07:54,190 perfect with that nomenclature. 149 00:07:54,190 --> 00:07:58,110 So these are the building blocks for DNA, for polymerization. 150 00:07:58,110 --> 00:08:03,450 And I just showed you how you grow from the 5 prime end-- 151 00:08:03,450 --> 00:08:06,420 that number's not shown there, but it's shown over here-- 152 00:08:06,420 --> 00:08:09,560 and you add to the 3 prime end. 153 00:08:09,560 --> 00:08:11,160 And the convention, remember, when 154 00:08:11,160 --> 00:08:14,970 we describe a strand of DNA, because we make it 5 prime 155 00:08:14,970 --> 00:08:18,480 to 3 prime, we write it 5 prime to 3 prime 156 00:08:18,480 --> 00:08:19,980 just so that we're all consistent 157 00:08:19,980 --> 00:08:21,840 and we know what we're talking about. 158 00:08:21,840 --> 00:08:26,070 Now I want to talk about two experiments that 159 00:08:26,070 --> 00:08:32,190 enlist the use of isotopes because these were quite useful 160 00:08:32,190 --> 00:08:37,980 early on to describe some of the characteristics of replication, 161 00:08:37,980 --> 00:08:41,370 so some of the details of replication, and also the fact 162 00:08:41,370 --> 00:08:44,430 that DNA was the genetic material. 163 00:08:44,430 --> 00:08:47,670 So isotopes are elements that share 164 00:08:47,670 --> 00:08:50,950 the same number of protons and electrons, 165 00:08:50,950 --> 00:08:53,910 but they differ in the number of neutrons. 166 00:08:53,910 --> 00:08:57,300 So the common isotopes of the elements 167 00:08:57,300 --> 00:09:01,380 that are in all the covalent structures of the body 168 00:09:01,380 --> 00:09:08,580 are hydrogen, carbon, and what I'm 169 00:09:08,580 --> 00:09:12,270 putting as the number next to it is the common isotope, 170 00:09:12,270 --> 00:09:17,610 so that number designates the sum of the number of neutrons 171 00:09:17,610 --> 00:09:18,480 and protons. 172 00:09:18,480 --> 00:09:21,300 So carbon-12, phosphorus-31. 173 00:09:24,860 --> 00:09:26,810 I should have put nitrogen here. 174 00:09:26,810 --> 00:09:27,337 Let's see. 175 00:09:27,337 --> 00:09:27,920 Wait a minute. 176 00:09:27,920 --> 00:09:30,800 I'm going to put these in order because it makes more sense. 177 00:09:30,800 --> 00:09:39,830 Phosphorus-31, nitrogen-14, PS, phosphorus-31, and sulfur. 178 00:09:39,830 --> 00:09:41,360 What's the sulfur isotope? 179 00:09:41,360 --> 00:09:43,670 32. 180 00:09:43,670 --> 00:09:47,660 So these are the common isotopes that 181 00:09:47,660 --> 00:09:51,770 comprise the majority of those elements within the body. 182 00:09:51,770 --> 00:09:53,390 But there are lots of experiments 183 00:09:53,390 --> 00:09:57,340 done that have different isotopes of these atoms 184 00:09:57,340 --> 00:10:08,180 that we can use as traces or markers, kind 185 00:10:08,180 --> 00:10:08,970 of the same thing. 186 00:10:08,970 --> 00:10:12,050 But you'll see when I mention the word tracer, to me, 187 00:10:12,050 --> 00:10:15,530 it brings up the radioactive isotopes because we 188 00:10:15,530 --> 00:10:17,370 use very little of them. 189 00:10:17,370 --> 00:10:19,910 So when we talk about these elements, 190 00:10:19,910 --> 00:10:23,420 there are different isotopes that we use commonly. 191 00:10:23,420 --> 00:10:26,570 There's the hydrogen that has an extra neutron. 192 00:10:26,570 --> 00:10:28,280 That's also called deuterium. 193 00:10:34,390 --> 00:10:38,680 And then there's the hydrogen that is radioactive. 194 00:10:38,680 --> 00:10:39,910 It's metastable. 195 00:10:39,910 --> 00:10:43,990 It will decay and emit a radioactive particle. 196 00:10:43,990 --> 00:10:48,460 So this one, we would call it the heavy isotope, 197 00:10:48,460 --> 00:10:54,990 and this one we would call the radioactive isotope. 198 00:10:54,990 --> 00:10:59,820 So we could trace certain hydrogens in biomolecules 199 00:10:59,820 --> 00:11:02,670 by either making those hydrogens the heavier ones, 200 00:11:02,670 --> 00:11:05,010 or making them the radioactive ones. 201 00:11:05,010 --> 00:11:07,980 Carbon also has useful isotopes. 202 00:11:07,980 --> 00:11:15,320 Carbon-13 is the heavy isotope and carbon-14 203 00:11:15,320 --> 00:11:17,250 is the radioactive isotope. 204 00:11:17,250 --> 00:11:19,380 So they're used quite commonly. 205 00:11:19,380 --> 00:11:22,470 And then things get a little bit different for nitrogen. 206 00:11:22,470 --> 00:11:26,790 We use a heavy isotope of nitrogen, which is N-15, just 207 00:11:26,790 --> 00:11:28,390 one extra neutron. 208 00:11:28,390 --> 00:11:31,830 But while there is a radioactive isotope of nitrogen, 209 00:11:31,830 --> 00:11:35,160 it's too short of a half-life to work within the lab. 210 00:11:35,160 --> 00:11:37,890 It's not useful to us, so we never talk about it. 211 00:11:37,890 --> 00:11:40,560 But I will describe to you an experiment 212 00:11:40,560 --> 00:11:43,890 with the heavy N-15 when we talk about the mechanism 213 00:11:43,890 --> 00:11:45,310 of replication. 214 00:11:45,310 --> 00:11:49,410 And then for phosphorus and sulfur, 215 00:11:49,410 --> 00:11:53,550 the most important ones are P-32, 216 00:11:53,550 --> 00:11:56,580 which is a radioactive phosphorus, 217 00:11:56,580 --> 00:12:02,550 and S-35, which is a radioactive sulfur. 218 00:12:02,550 --> 00:12:06,210 There is another radioactive phosphorus that's P-33. 219 00:12:06,210 --> 00:12:07,800 Has a slightly longer half life. 220 00:12:07,800 --> 00:12:09,540 It's kind of handy to work with if you 221 00:12:09,540 --> 00:12:11,490 have certain experiments. 222 00:12:11,490 --> 00:12:16,050 The half-lives vary a lot, but the half-life of tritium C-14 223 00:12:16,050 --> 00:12:17,130 are long. 224 00:12:17,130 --> 00:12:19,140 That's why we do carbon dating. 225 00:12:19,140 --> 00:12:25,950 The half-lives of P-32 are short, less daytime frame, 226 00:12:25,950 --> 00:12:27,870 and sulfur 35 is a little bit longer. 227 00:12:27,870 --> 00:12:30,810 But those are nuances that you don't need to worry about. 228 00:12:30,810 --> 00:12:33,210 Now how are these isotopes useful 229 00:12:33,210 --> 00:12:38,280 as traces and markers to tell us about biology and details 230 00:12:38,280 --> 00:12:39,430 of biology? 231 00:12:39,430 --> 00:12:41,160 So what I'm going to just show you here 232 00:12:41,160 --> 00:12:43,290 is a particular experiment that's 233 00:12:43,290 --> 00:12:46,350 carried out with what are known as baculovirus. 234 00:12:46,350 --> 00:12:50,340 So viruses infect eukaryotic cells. 235 00:12:50,340 --> 00:12:53,220 Baculoviruses infect bacteria. 236 00:12:53,220 --> 00:12:55,260 So there's a commonality there. 237 00:12:55,260 --> 00:12:59,550 And when baculoviruses infect bacteria, just as the parallel 238 00:12:59,550 --> 00:13:03,540 with eukaryotic cells, they deposit material 239 00:13:03,540 --> 00:13:06,670 into the bacteria so they can replicate, 240 00:13:06,670 --> 00:13:12,120 so they can hijack the bacteria to make new baculoviruses. 241 00:13:12,120 --> 00:13:15,300 So an early experiment was done to ask 242 00:13:15,300 --> 00:13:19,770 what is the genetic material that transfers the information 243 00:13:19,770 --> 00:13:23,820 from the baculovirus to the bacterial cell 244 00:13:23,820 --> 00:13:26,640 in order to make more baculovirus. 245 00:13:26,640 --> 00:13:30,780 So they were able to demonstrate, in this Hershey 246 00:13:30,780 --> 00:13:34,440 and Chase experiment, that you could 247 00:13:34,440 --> 00:13:40,830 label protein and DNA with particular isotopes 248 00:13:40,830 --> 00:13:44,220 and find out what part of the baculovirus 249 00:13:44,220 --> 00:13:47,220 was important for transferring information 250 00:13:47,220 --> 00:13:50,340 for the production of new baculovirus. 251 00:13:50,340 --> 00:13:53,800 So they wanted to label the capsid protein, 252 00:13:53,800 --> 00:13:56,130 so the proteinaceous material that's 253 00:13:56,130 --> 00:13:58,980 on the outside of this lunar lander. 254 00:13:58,980 --> 00:14:00,180 This is a baculovirus. 255 00:14:00,180 --> 00:14:02,280 It's sort of amazing how it looks. 256 00:14:02,280 --> 00:14:04,230 So they wanted to label the protein. 257 00:14:04,230 --> 00:14:07,230 But they also wanted to label the contents 258 00:14:07,230 --> 00:14:12,640 of the baculovirus, the DNA, with tracer radioisotopes. 259 00:14:12,640 --> 00:14:14,460 So what would be the best isotopes 260 00:14:14,460 --> 00:14:18,360 to use if you wanted to differentiate between protein 261 00:14:18,360 --> 00:14:20,340 and nucleic acid? 262 00:14:20,340 --> 00:14:22,030 And what you want to think about is, 263 00:14:22,030 --> 00:14:24,840 what does protein have that nucleic acids don't have, 264 00:14:24,840 --> 00:14:27,660 and what do nucleic acids have that proteins don't have? 265 00:14:27,660 --> 00:14:31,870 Which are the elements that are important for differentiating? 266 00:14:31,870 --> 00:14:32,370 Yeah. 267 00:14:32,370 --> 00:14:34,885 Over there. 268 00:14:34,885 --> 00:14:35,760 AUDIENCE: Phosphorus. 269 00:14:35,760 --> 00:14:37,218 BARBARA IMPERIALI: Yes, phosphorus. 270 00:14:37,218 --> 00:14:40,232 Do you want to give a little bit more of a-- 271 00:14:40,232 --> 00:14:44,230 AUDIENCE: [INAUDIBLE] 272 00:14:44,230 --> 00:14:45,270 BARBARA IMPERIALI: OK. 273 00:14:45,270 --> 00:14:48,210 So the answer is we'd use phosphorus 274 00:14:48,210 --> 00:14:52,740 to label nucleic acids because that phosphodiester backbone is 275 00:14:52,740 --> 00:14:54,180 rich in phosphorus. 276 00:14:54,180 --> 00:14:56,250 There are a few phosphates in proteins, 277 00:14:56,250 --> 00:14:57,450 but they're very transient. 278 00:14:57,450 --> 00:14:58,770 They're part of signaling. 279 00:14:58,770 --> 00:15:02,190 But every nucleotide has a phosphorus 280 00:15:02,190 --> 00:15:03,640 in every building block. 281 00:15:03,640 --> 00:15:07,080 And then in proteins, they have sulfur in them 282 00:15:07,080 --> 00:15:09,270 where nucleic acids do not. 283 00:15:09,270 --> 00:15:13,410 And the sulfur is in two amino acids cystine and methionine. 284 00:15:13,410 --> 00:15:17,720 So you can use those two tracers for the building blocks. 285 00:15:17,720 --> 00:15:20,010 And the way the experiment works is 286 00:15:20,010 --> 00:15:25,080 if you've labeled the protein with sulfur, you infect a cell, 287 00:15:25,080 --> 00:15:27,390 the virus replicates. 288 00:15:27,390 --> 00:15:30,240 And what you're going to do is then 289 00:15:30,240 --> 00:15:35,550 centrifuge the bacterial cells to see what's in the cell, 290 00:15:35,550 --> 00:15:39,360 and then you can, alternatively, label the DNA-- 291 00:15:39,360 --> 00:15:41,100 and in this case, I've shown it as green, 292 00:15:41,100 --> 00:15:43,110 but that would be the other isotope-- 293 00:15:43,110 --> 00:15:46,290 let it infect the cell and deposit its contents 294 00:15:46,290 --> 00:15:49,190 in the cell, centrifuge the cells out. 295 00:15:49,190 --> 00:15:52,260 And you want to know where the radioactivity is, 296 00:15:52,260 --> 00:15:54,090 and the radioactivity is strictly 297 00:15:54,090 --> 00:15:59,010 associated with phosphorus-32 because the genetic material 298 00:15:59,010 --> 00:16:02,370 that coded for the production of new baculovirus 299 00:16:02,370 --> 00:16:04,080 is the thing that stays associated 300 00:16:04,080 --> 00:16:05,770 with the bacterial cell. 301 00:16:05,770 --> 00:16:11,510 In contrast, there's no radioactivity associated when 302 00:16:11,510 --> 00:16:14,960 you label the cells with S-35. 303 00:16:14,960 --> 00:16:15,560 All right? 304 00:16:15,560 --> 00:16:17,030 Nice experiment. 305 00:16:17,030 --> 00:16:18,860 Easy way to do it. 306 00:16:18,860 --> 00:16:20,663 Rather nice way to do it. 307 00:16:20,663 --> 00:16:22,580 The other experiment I want to describe to you 308 00:16:22,580 --> 00:16:25,460 very briefly because it relies on central irrigation 309 00:16:25,460 --> 00:16:28,550 technology that's very powerful. 310 00:16:28,550 --> 00:16:37,980 And it's a method that utilizes N-15 very, very nicely. 311 00:16:37,980 --> 00:16:40,350 So let me tell you about that experiment. 312 00:16:40,350 --> 00:16:42,210 There are things in the laboratory 313 00:16:42,210 --> 00:16:44,970 that we use every day, ultra centrifuges 314 00:16:44,970 --> 00:16:48,480 and regular centrifuges, that spin at a very fast speed 315 00:16:48,480 --> 00:16:50,640 where we can really differentiate things 316 00:16:50,640 --> 00:16:55,710 by molecular mass, which relates to sedimentation coefficients. 317 00:16:55,710 --> 00:16:59,880 How fast does the particle spin to the bottom of the tube 318 00:16:59,880 --> 00:17:02,620 when it's under high centrifugal force? 319 00:17:02,620 --> 00:17:05,819 The heavier it is, the faster it will spin. 320 00:17:05,819 --> 00:17:08,190 So the question that came up very early on-- it 321 00:17:08,190 --> 00:17:09,780 looks like a nonsense question now 322 00:17:09,780 --> 00:17:13,920 because it looks so obvious that we replicate DNA the way we do. 323 00:17:13,920 --> 00:17:16,920 But originally, it wasn't absolutely certain 324 00:17:16,920 --> 00:17:20,640 whether DNA was replicated through a semi-conservative 325 00:17:20,640 --> 00:17:23,160 mechanism, where the strands came apart 326 00:17:23,160 --> 00:17:26,069 and you made two copies, a copy of each strand, 327 00:17:26,069 --> 00:17:31,260 to make two identical daughter new double-stranded DNA. 328 00:17:31,260 --> 00:17:33,720 Alternatively, could you have a mechanism that 329 00:17:33,720 --> 00:17:35,880 was quite conservative, you kept your DNA, 330 00:17:35,880 --> 00:17:38,170 and somehow you made a copy of it. 331 00:17:38,170 --> 00:17:39,930 A little harder for me to understand. 332 00:17:39,930 --> 00:17:42,870 So your two new strands, one would 333 00:17:42,870 --> 00:17:47,100 look like the original one, but one would be very different. 334 00:17:47,100 --> 00:17:50,070 Or dispersive, where you're just copying bits of the DNA 335 00:17:50,070 --> 00:17:54,270 and somehow reassembling this gigantic jigsaw puzzle. 336 00:17:54,270 --> 00:17:57,630 The centrifugation experiment allowed 337 00:17:57,630 --> 00:18:00,180 people to absolutely and clearly state 338 00:18:00,180 --> 00:18:04,490 that the replication is via a semi-conservative process. 339 00:18:04,490 --> 00:18:05,690 So let's walk through it. 340 00:18:05,690 --> 00:18:10,470 And what was done is that the nucleotides within the DNA 341 00:18:10,470 --> 00:18:13,290 were all labeled with heavy nitrogen. 342 00:18:13,290 --> 00:18:15,400 So for every nucleic acid-- 343 00:18:15,400 --> 00:18:17,130 let's say, for example, when there's 344 00:18:17,130 --> 00:18:21,540 an adenine in the backbone, there are five nitrogens, 345 00:18:21,540 --> 00:18:23,520 so that would be five atomic mass 346 00:18:23,520 --> 00:18:28,680 units heavier than nitrogen-14 in that nucleobase. 347 00:18:28,680 --> 00:18:32,790 So N-15 nucleobase, five mass units heavier than N-14 348 00:18:32,790 --> 00:18:33,630 nucleobase. 349 00:18:33,630 --> 00:18:35,130 Make sense to everybody? 350 00:18:35,130 --> 00:18:35,630 All right. 351 00:18:35,630 --> 00:18:37,520 So it's heavier by some amount. 352 00:18:37,520 --> 00:18:39,480 It's not a massive amount, but it's 353 00:18:39,480 --> 00:18:42,870 enough to differentiate in a centrifugation experiment. 354 00:18:42,870 --> 00:18:46,680 So the bacteria were first grown up in heavy nitrogen, 355 00:18:46,680 --> 00:18:53,160 so all of the DNA sediments at a certain rate and place. 356 00:18:53,160 --> 00:18:56,040 And it's all got N-15 in it, so it's as heavy 357 00:18:56,040 --> 00:18:57,330 as it can sediment. 358 00:18:57,330 --> 00:19:00,150 And then they let the bacteria replicate 359 00:19:00,150 --> 00:19:05,350 in the presence of N-14, the lighter isotope of nitrogen. 360 00:19:05,350 --> 00:19:07,470 And what one would anticipate to happen 361 00:19:07,470 --> 00:19:09,600 is whenever you replicate, you peel apart 362 00:19:09,600 --> 00:19:14,670 the two heavy strands, and each one pairs with a light strand. 363 00:19:14,670 --> 00:19:20,220 So you now have two copies, two new identical copies of DNA 364 00:19:20,220 --> 00:19:27,160 where half of one strand has N-15, one strand has N-14. 365 00:19:27,160 --> 00:19:30,420 So it will sediment less quickly because it 366 00:19:30,420 --> 00:19:32,250 is of a different density, a lighter 367 00:19:32,250 --> 00:19:34,530 density than the all N-15. 368 00:19:34,530 --> 00:19:37,680 So you would get an intermediate weight band 369 00:19:37,680 --> 00:19:40,560 in the centrifuge tube when you're sedimenting. 370 00:19:40,560 --> 00:19:44,130 If you peel those two strands apart again, 371 00:19:44,130 --> 00:19:46,020 you've got a heavy and a light. 372 00:19:46,020 --> 00:19:48,210 If you're growing in N-14, the light 373 00:19:48,210 --> 00:19:54,090 will combine with another light, and there are two of those. 374 00:19:54,090 --> 00:19:56,070 And then the heavy will combine with a light. 375 00:19:56,070 --> 00:19:59,220 So you'll get new sedimentation where you still 376 00:19:59,220 --> 00:20:01,500 have a band that's the combination 377 00:20:01,500 --> 00:20:03,300 of the light and heavy. 378 00:20:03,300 --> 00:20:05,790 But then you start having some material 379 00:20:05,790 --> 00:20:07,330 that is all of the light. 380 00:20:07,330 --> 00:20:09,180 So this would be this cycle. 381 00:20:09,180 --> 00:20:13,800 So two that are still light plus heavy, and then two that 382 00:20:13,800 --> 00:20:15,930 are exclusively light. 383 00:20:15,930 --> 00:20:17,730 You keep on replicating, and you'll 384 00:20:17,730 --> 00:20:19,590 keep on diluting the heavy. 385 00:20:19,590 --> 00:20:22,300 Does that experiment make sense? 386 00:20:22,300 --> 00:20:23,640 It's kind of a cool experiment. 387 00:20:23,640 --> 00:20:25,500 You can hardly believe it's feasible, 388 00:20:25,500 --> 00:20:29,100 but the centrifugation ability to differentiate 389 00:20:29,100 --> 00:20:31,320 with these isotopes is really valuable. 390 00:20:31,320 --> 00:20:34,680 So I just want to put in a plug for the use of isotopes. 391 00:20:34,680 --> 00:20:36,930 Obviously, they're used in nuclear physics. 392 00:20:36,930 --> 00:20:41,310 We use a lot of the radioactive isotopes for different reasons. 393 00:20:41,310 --> 00:20:44,340 But in biology, they are indispensable for some 394 00:20:44,340 --> 00:20:45,360 of these experiments. 395 00:20:45,360 --> 00:20:47,250 And even to this day, you can do things 396 00:20:47,250 --> 00:20:52,410 with isotopes you can't by other experiments, either 397 00:20:52,410 --> 00:20:54,410 the radioactive or the heavy. 398 00:20:54,410 --> 00:20:58,830 There's a great deal of work done nowadays in proteomics 399 00:20:58,830 --> 00:21:01,830 using mass spectrometry and heavy isotopes, 400 00:21:01,830 --> 00:21:04,680 where you can really track where things go 401 00:21:04,680 --> 00:21:09,030 and treat cancer cells differently from healthy cells, 402 00:21:09,030 --> 00:21:10,770 but actually track what's happening 403 00:21:10,770 --> 00:21:14,715 by putting heavy isotopes in one of the growing dishes of cells. 404 00:21:19,350 --> 00:21:22,890 So I like both of those experiments a lot. 405 00:21:22,890 --> 00:21:26,100 I would put them in the category of oldies but goodies. 406 00:21:26,100 --> 00:21:26,760 All right. 407 00:21:26,760 --> 00:21:29,100 Now I've got some details over here 408 00:21:29,100 --> 00:21:32,915 that we've got some explaining to do as we move forward. 409 00:21:32,915 --> 00:21:34,290 And I just want to, first of all, 410 00:21:34,290 --> 00:21:36,540 start with a couple of details that I want 411 00:21:36,540 --> 00:21:40,020 to highlight on this board. 412 00:21:43,630 --> 00:21:51,100 First of all, prokaryotes such as bacteria, E. Coli, 413 00:21:51,100 --> 00:21:53,620 and eukaryotes such as human cells. 414 00:21:53,620 --> 00:21:56,330 Have some differences in their DNA. 415 00:21:56,330 --> 00:21:59,200 So the size of the bacterial genome 416 00:21:59,200 --> 00:22:02,410 is about five million base pairs. 417 00:22:02,410 --> 00:22:06,310 The size of the human genome is not quite 1,000 times bigger, 418 00:22:06,310 --> 00:22:10,570 but a good deal bigger. 419 00:22:10,570 --> 00:22:15,640 The DNA in bacteria is circular, whereas the DNA 420 00:22:15,640 --> 00:22:18,220 in eukaryotic cells is linear. 421 00:22:18,220 --> 00:22:21,370 And it's actually in the form-- you see all these little images 422 00:22:21,370 --> 00:22:25,210 where you see the pairs of chromosomes sort of stuck 423 00:22:25,210 --> 00:22:27,400 together at the center, and that's 424 00:22:27,400 --> 00:22:30,160 linear DNA from one end to the other. 425 00:22:30,160 --> 00:22:33,500 In bacteria, the DNA is circular, 426 00:22:33,500 --> 00:22:34,750 so it doesn't have an end. 427 00:22:34,750 --> 00:22:36,850 So they're different, all right? 428 00:22:36,850 --> 00:22:40,300 So replicating circular DNA is a little bit 429 00:22:40,300 --> 00:22:44,620 different from replicating linear DNA. 430 00:22:44,620 --> 00:22:48,970 Now in both cases, the DNA has to be packaged up 431 00:22:48,970 --> 00:22:50,960 so it will fit in the cell. 432 00:22:50,960 --> 00:22:53,930 Otherwise, it's just too much of a disordered thing. 433 00:22:53,930 --> 00:22:59,500 In the case of bacteria, the circular DNA is wrapped up 434 00:22:59,500 --> 00:23:01,000 and super coiled with what are known 435 00:23:01,000 --> 00:23:04,240 as a polyamines, compounds that are very positively 436 00:23:04,240 --> 00:23:09,190 charged to neutralize all that negative charge in the DNA. 437 00:23:09,190 --> 00:23:13,240 And in man and other eukaryotes, the chromosomes 438 00:23:13,240 --> 00:23:16,240 are wrapped around very, very positively charged 439 00:23:16,240 --> 00:23:18,670 proteins known as histones. 440 00:23:18,670 --> 00:23:22,540 They're similar principles, but they're different entities. 441 00:23:22,540 --> 00:23:26,440 So when we start talking about replication 442 00:23:26,440 --> 00:23:31,150 of a prokaryotic cell, here's the typical circular DNA. 443 00:23:31,150 --> 00:23:33,340 So you can see the double-stranded DNA. 444 00:23:33,340 --> 00:23:38,570 And I've put a little symbol here that I call the ORI. 445 00:23:42,800 --> 00:23:47,480 So that's origin of initiation. 446 00:23:50,360 --> 00:23:54,295 So that is the place where you start copying your DNA. 447 00:23:54,295 --> 00:23:55,670 And we're going to talk about how 448 00:23:55,670 --> 00:23:59,760 to spot these places in genomes in a moment. 449 00:23:59,760 --> 00:24:05,720 And so the bacterial genome is copied bidirectionally, 450 00:24:05,720 --> 00:24:10,880 so you can go in both directions copying in the appropriate 451 00:24:10,880 --> 00:24:13,780 direction-- we'll talk about that in a moment-- 452 00:24:13,780 --> 00:24:19,070 to make the entire circular DNA and a perfect copy of the DNA 453 00:24:19,070 --> 00:24:21,470 with bidirectional copying. 454 00:24:21,470 --> 00:24:23,810 And that's not bad for small genomes 455 00:24:23,810 --> 00:24:26,150 because you've only got one origin of replication. 456 00:24:26,150 --> 00:24:30,050 You've got to get all the way around the circular DNA. 457 00:24:30,050 --> 00:24:32,630 It's quite a bit smaller than the human one. 458 00:24:32,630 --> 00:24:37,310 But what do you do with gigantic genomes 459 00:24:37,310 --> 00:24:40,520 such as the one that's about 1,000 times bigger 460 00:24:40,520 --> 00:24:42,170 than the bacterial one? 461 00:24:42,170 --> 00:24:45,740 How do you manage to still catalyze 462 00:24:45,740 --> 00:24:48,020 the replication of the entire genome 463 00:24:48,020 --> 00:24:51,290 quickly enough to make the copy of DNA 464 00:24:51,290 --> 00:24:53,810 in some reasonable amount of time? 465 00:24:53,810 --> 00:24:56,930 And that is taking also into consideration 466 00:24:56,930 --> 00:24:59,690 that the speed of bacterial replication 467 00:24:59,690 --> 00:25:02,210 is 1,000 base pairs per second. 468 00:25:02,210 --> 00:25:05,840 Pretty impressive-- 1,000 of those bonds made every second-- 469 00:25:05,840 --> 00:25:08,150 whereas in eukaryotes, it sits somewhere down 470 00:25:08,150 --> 00:25:12,650 from 50 base pairs per second, depending on conditions. 471 00:25:12,650 --> 00:25:15,400 These are not very explicit numbers. 472 00:25:15,400 --> 00:25:18,590 There's a little bit of variability. 473 00:25:18,590 --> 00:25:19,840 So how would you do it? 474 00:25:19,840 --> 00:25:23,470 If you had this massive chunk of gene, 475 00:25:23,470 --> 00:25:28,540 and you've got to copy it just really pretty quickly in order 476 00:25:28,540 --> 00:25:32,530 to replicate an entire cell contents in eight hours, 477 00:25:32,530 --> 00:25:33,820 what would you do? 478 00:25:33,820 --> 00:25:36,461 How could you expedite things? 479 00:25:36,461 --> 00:25:38,870 Yeah. 480 00:25:38,870 --> 00:25:40,280 Yes. 481 00:25:40,280 --> 00:25:43,740 So the answer here is start in a lot of different places 482 00:25:43,740 --> 00:25:45,890 so there's a lot of collaborative work going 483 00:25:45,890 --> 00:25:50,330 on all along that large genome. 484 00:25:50,330 --> 00:25:55,480 And so when the replication of the long linear chromosomes 485 00:25:55,480 --> 00:25:57,490 that are in eukaryotic cells, you 486 00:25:57,490 --> 00:26:01,420 end up with just a lot of origins of replication. 487 00:26:01,420 --> 00:26:03,880 So you're basically starting all over the place 488 00:26:03,880 --> 00:26:06,520 so you can get the job done quickly enough for it 489 00:26:06,520 --> 00:26:07,460 to make sense. 490 00:26:07,460 --> 00:26:10,910 Does that make sense to everybody? 491 00:26:10,910 --> 00:26:12,460 So start in a lot of places. 492 00:26:12,460 --> 00:26:13,820 It's a pretty easy thing. 493 00:26:13,820 --> 00:26:14,320 All right. 494 00:26:14,320 --> 00:26:21,670 Now I mentioned very briefly that before you can copy DNA, 495 00:26:21,670 --> 00:26:23,470 you have to unpack it. 496 00:26:23,470 --> 00:26:27,940 Now we think a lot about packaging DNA, 497 00:26:27,940 --> 00:26:29,630 less about unpacking it. 498 00:26:29,630 --> 00:26:32,660 So I've got these pictures sort of in a reverse direction. 499 00:26:32,660 --> 00:26:37,240 So it's only this form of DNA stretched out 500 00:26:37,240 --> 00:26:41,290 that can be copied, whereas the DNA in your cells 501 00:26:41,290 --> 00:26:44,530 is bundled up very tightly into chromosomes. 502 00:26:44,530 --> 00:26:46,360 How are they put together? 503 00:26:46,360 --> 00:26:50,230 Those chromosomes are made up of chromatin, 504 00:26:50,230 --> 00:26:54,400 which is a bunch of balls which are histone proteins with DNA 505 00:26:54,400 --> 00:26:57,580 wrapped around them. 506 00:26:57,580 --> 00:26:59,950 That's compact chromatin. 507 00:26:59,950 --> 00:27:00,590 Sorry, guys. 508 00:27:00,590 --> 00:27:03,850 Let me go back, not to go forwards. 509 00:27:03,850 --> 00:27:07,030 So this is chromatin in a compact form. 510 00:27:07,030 --> 00:27:09,370 This is chromatin now unraveled. 511 00:27:09,370 --> 00:27:11,410 They look more like beads on a string, 512 00:27:11,410 --> 00:27:14,410 DNA wrapped around each histone protein 513 00:27:14,410 --> 00:27:17,440 to form those nucleosomes structures. 514 00:27:17,440 --> 00:27:21,050 And then each nucleosome looks like this. 515 00:27:21,050 --> 00:27:24,680 It's got protein in the middle and DNA wrapped around it. 516 00:27:24,680 --> 00:27:27,430 So in order to go forward and replicate, 517 00:27:27,430 --> 00:27:30,100 we have to unpackage DNA. 518 00:27:30,100 --> 00:27:33,280 There are lots of signals to unpackage the DNA that we 519 00:27:33,280 --> 00:27:35,050 will talk about later. 520 00:27:35,050 --> 00:27:37,610 The nucleosomes look like this. 521 00:27:37,610 --> 00:27:42,585 So you can trace the DNA and the proteins bundled in the middle. 522 00:27:42,585 --> 00:27:43,960 And if we have time at the end, I 523 00:27:43,960 --> 00:27:48,610 will show you the video of the packaging of DNA 524 00:27:48,610 --> 00:27:51,100 and the video of the replication of DNA. 525 00:27:51,100 --> 00:27:52,870 They're about a minute and a half each, 526 00:27:52,870 --> 00:27:54,280 so it's not a whole bunch. 527 00:27:54,280 --> 00:27:56,830 Later on, we will talk about what 528 00:27:56,830 --> 00:28:00,280 happens when there is a determination that a cell needs 529 00:28:00,280 --> 00:28:02,320 to replicate its genome. 530 00:28:02,320 --> 00:28:05,110 A bunch of things have to happen as signals 531 00:28:05,110 --> 00:28:07,150 to do the unwrapping. 532 00:28:07,150 --> 00:28:09,160 And one of those things is actually 533 00:28:09,160 --> 00:28:13,150 to alter the charged state of the histone proteins 534 00:28:13,150 --> 00:28:15,670 so you neutralize the positive charges 535 00:28:15,670 --> 00:28:17,620 so the DNA can unravel for it. 536 00:28:17,620 --> 00:28:18,190 Makes sense. 537 00:28:18,190 --> 00:28:21,370 It's still just a plain old electrostatic interaction. 538 00:28:21,370 --> 00:28:25,180 So the synthesis of DNA is what's 539 00:28:25,180 --> 00:28:35,530 known as template-driven polymerization. 540 00:28:42,740 --> 00:28:43,240 OK. 541 00:28:43,240 --> 00:28:46,380 So template-driven polymerization. 542 00:28:46,380 --> 00:28:49,210 You'll get a sense of exactly what that means. 543 00:28:49,210 --> 00:28:51,850 We know the two strands are complementary, 544 00:28:51,850 --> 00:28:53,980 and we're going to use one strand as the code 545 00:28:53,980 --> 00:28:56,560 to make a new complementary strand. 546 00:28:56,560 --> 00:28:59,920 So when we're making DNA, we have one strand there. 547 00:28:59,920 --> 00:29:02,950 That's what's known as the template strand. 548 00:29:02,950 --> 00:29:06,640 I want to make a complementary copy of that strand. 549 00:29:06,640 --> 00:29:09,880 So what the DNA polymerase is going to do 550 00:29:09,880 --> 00:29:15,190 is systematically add nucleotides 551 00:29:15,190 --> 00:29:20,140 to the 3 prime OH of the ribose, and then you keep growing. 552 00:29:20,140 --> 00:29:22,660 But the base that gets put in is the one 553 00:29:22,660 --> 00:29:26,560 that's complementary to the base on the template strand. 554 00:29:26,560 --> 00:29:29,100 So if it's thiamine on the template strand, 555 00:29:29,100 --> 00:29:34,300 it's adenine put in on the new strand, and so on. 556 00:29:34,300 --> 00:29:36,310 And just to get these numbers again, 557 00:29:36,310 --> 00:29:40,930 the new strand is grown from 5 prime to 3 prime. 558 00:29:40,930 --> 00:29:45,180 The old strand is read 3 prime to 5 prime. 559 00:29:45,180 --> 00:29:48,550 So you're reading a strand and you're reading it 3 to 5, 560 00:29:48,550 --> 00:29:50,590 and you're making 5 to 3. 561 00:29:50,590 --> 00:29:53,800 That's how you end up with complementary anti-parallel 562 00:29:53,800 --> 00:29:55,600 strands. 563 00:29:55,600 --> 00:29:58,080 OK with that, everyone? 564 00:29:58,080 --> 00:29:58,690 All right. 565 00:29:58,690 --> 00:30:01,420 And when you form the bond, you go in 566 00:30:01,420 --> 00:30:07,180 with nucleoside triphosphates, you form a new phosphodiester 567 00:30:07,180 --> 00:30:09,330 with just one of those phosphoruses, 568 00:30:09,330 --> 00:30:11,860 and you kick out phosphate phosphate, 569 00:30:11,860 --> 00:30:16,700 which we would call pyrophosphate or diphosphate. 570 00:30:16,700 --> 00:30:17,200 OK. 571 00:30:17,200 --> 00:30:19,160 So here you could just sort of see, just 572 00:30:19,160 --> 00:30:21,410 check that you know what you're doing. 573 00:30:21,410 --> 00:30:24,140 I'm going to just say this is really a pretty simple thing. 574 00:30:24,140 --> 00:30:26,960 We're going to put in a T, then we're going to put in an a 575 00:30:26,960 --> 00:30:30,380 then we're going to put in a C because our template is telling 576 00:30:30,380 --> 00:30:34,910 us to put in the appropriate purine or pyrimidine that forms 577 00:30:34,910 --> 00:30:36,770 a nice base pair with that. 578 00:30:36,770 --> 00:30:39,980 So there's a question here. 579 00:30:39,980 --> 00:30:42,330 But I've already given you the answer to it. 580 00:30:42,330 --> 00:30:44,690 So it should be very straightforward 581 00:30:44,690 --> 00:30:48,230 for you to look at a template strand 582 00:30:48,230 --> 00:30:51,710 and decide what would be on the complementary strand, 583 00:30:51,710 --> 00:30:54,410 and also decide on the directionality 584 00:30:54,410 --> 00:30:57,970 of the complementary strand. 585 00:30:57,970 --> 00:31:00,070 Now origins of replication. 586 00:31:00,070 --> 00:31:03,760 What do they look like? 587 00:31:03,760 --> 00:31:05,840 It's the old where do I start problem. 588 00:31:05,840 --> 00:31:06,623 Where do I start? 589 00:31:06,623 --> 00:31:07,915 What's the best place to start? 590 00:31:10,470 --> 00:31:12,610 Someone? 591 00:31:12,610 --> 00:31:13,480 Not you. 592 00:31:13,480 --> 00:31:15,050 Not you. 593 00:31:15,050 --> 00:31:17,110 Anyone else want to answer here? 594 00:31:17,110 --> 00:31:18,293 I've got this whole genome. 595 00:31:18,293 --> 00:31:19,210 I've got to get going. 596 00:31:19,210 --> 00:31:20,860 I've got to start making a copy of it. 597 00:31:20,860 --> 00:31:26,170 Where's the best place to break into it to start making copies? 598 00:31:26,170 --> 00:31:26,730 Yeah. 599 00:31:26,730 --> 00:31:29,130 There. 600 00:31:29,130 --> 00:31:31,460 AUDIENCE: [INAUDIBLE] 601 00:31:31,460 --> 00:31:32,660 BARBARA IMPERIALI: Correct. 602 00:31:32,660 --> 00:31:33,290 It's simple. 603 00:31:33,290 --> 00:31:35,360 It's the difference between two and three. 604 00:31:35,360 --> 00:31:36,950 If you go to the area where there's 605 00:31:36,950 --> 00:31:41,210 lots of G's and C's, every GC has three hydrogen bonds. 606 00:31:41,210 --> 00:31:42,800 If you go to a different area where 607 00:31:42,800 --> 00:31:45,530 there happen to be a lot of A's and T's in a row, 608 00:31:45,530 --> 00:31:48,530 each pair is only worth two hydrogen bonds. 609 00:31:48,530 --> 00:31:51,260 You're going to pick the stretch that has the most 610 00:31:51,260 --> 00:31:54,980 A's and T's because it's the place where the base pairing is 611 00:31:54,980 --> 00:31:56,240 weaker. 612 00:31:56,240 --> 00:31:58,800 It's actually very simple concept. 613 00:31:58,800 --> 00:32:01,400 So it would be the AT-rich region 614 00:32:01,400 --> 00:32:03,860 is the place where origins of replication 615 00:32:03,860 --> 00:32:04,920 are more predominant. 616 00:32:04,920 --> 00:32:05,420 OK. 617 00:32:05,420 --> 00:32:07,010 So what we're going to do now-- 618 00:32:07,010 --> 00:32:09,110 and this is going to be tricky, but I'm 619 00:32:09,110 --> 00:32:11,000 going to make this work-- 620 00:32:11,000 --> 00:32:13,670 is we're going to talk about replicating 621 00:32:13,670 --> 00:32:15,730 an entire chunk of gene. 622 00:32:15,730 --> 00:32:20,810 And what I've done here is I've put the pieces that we're 623 00:32:20,810 --> 00:32:24,350 going to discuss over here. 624 00:32:24,350 --> 00:32:25,480 That's my menu. 625 00:32:25,480 --> 00:32:28,100 And we're going to work through what we need 626 00:32:28,100 --> 00:32:30,320 to copy a large chunk of DNA. 627 00:32:30,320 --> 00:32:31,280 All right? 628 00:32:31,280 --> 00:32:32,450 Everybody with me? 629 00:32:32,450 --> 00:32:35,690 So I redrew this this morning. 630 00:32:35,690 --> 00:32:37,070 I promised myself I'm going to be 631 00:32:37,070 --> 00:32:41,600 super tidy on this blackboard, which is a far stretch for me. 632 00:32:41,600 --> 00:32:44,895 So replication, we know where replication starts, 633 00:32:44,895 --> 00:32:47,030 AT-rich region. 634 00:32:47,030 --> 00:32:49,070 We could pick those out in the genome. 635 00:32:49,070 --> 00:32:53,720 We know that we've got to unwind the DNA to make a copy of it, 636 00:32:53,720 --> 00:32:57,150 so we must have had to unwrap the DNA beforehand. 637 00:32:57,150 --> 00:32:58,650 These are all known things. 638 00:32:58,650 --> 00:33:01,520 So let's now take a look at double-stranded DNA 639 00:33:01,520 --> 00:33:05,260 and try and figure out how we use all the components. 640 00:33:05,260 --> 00:33:08,660 Actually, I'm going to bring this board down because I 641 00:33:08,660 --> 00:33:12,260 don't see it as well up there. 642 00:33:12,260 --> 00:33:15,470 How do we make use of all these components 643 00:33:15,470 --> 00:33:17,300 that are part of the menu that we need 644 00:33:17,300 --> 00:33:19,860 to make the new strand of DNA? 645 00:33:19,860 --> 00:33:35,245 So we have double-stranded DNA and there are base pairs 646 00:33:35,245 --> 00:33:35,745 across. 647 00:33:39,180 --> 00:33:42,200 That's typical double-stranded DNA. 648 00:33:42,200 --> 00:33:45,110 That's what we're starting from, unwrapped. 649 00:33:45,110 --> 00:33:47,570 I haven't made it helical because my helices will 650 00:33:47,570 --> 00:33:48,530 get messy. 651 00:33:48,530 --> 00:33:49,580 And so we need to start. 652 00:33:49,580 --> 00:33:52,070 So the first thing that will happen 653 00:33:52,070 --> 00:33:55,550 is that the important enzymes like the helicase 654 00:33:55,550 --> 00:33:59,030 will scan to find an origin of replication, 655 00:33:59,030 --> 00:34:06,887 so somewhere for the process to start because that's where 656 00:34:06,887 --> 00:34:07,970 they're going to break in. 657 00:34:07,970 --> 00:34:12,770 So David, one of your TA's, works in Steve Bell's lab. 658 00:34:12,770 --> 00:34:15,170 And they study the mammalian origin 659 00:34:15,170 --> 00:34:18,860 of replication complex, which is this much more 660 00:34:18,860 --> 00:34:20,810 complicated situation than what I'm 661 00:34:20,810 --> 00:34:22,940 going to describe to you today. 662 00:34:22,940 --> 00:34:25,040 But they do fabulous single molecule 663 00:34:25,040 --> 00:34:29,393 work to show how those pieces are assembled. 664 00:34:29,393 --> 00:34:30,560 But I'll tell you the truth. 665 00:34:30,560 --> 00:34:32,270 As dorky as I am, the thing that I 666 00:34:32,270 --> 00:34:40,040 find the most cool about the origin of replication complex 667 00:34:40,040 --> 00:34:43,030 is that it spells ORC. 668 00:34:43,030 --> 00:34:45,260 And if any of you are Lord of the Rings 669 00:34:45,260 --> 00:34:50,239 fans, who can't be excited by a complex called the ORC complex? 670 00:34:50,239 --> 00:34:54,469 So there's going to be the screening 671 00:34:54,469 --> 00:34:56,239 of the genome for ORI. 672 00:34:56,239 --> 00:35:01,370 And I want you to remember what is up here 673 00:35:01,370 --> 00:35:03,510 is the rest of the chromosome. 674 00:35:03,510 --> 00:35:05,570 It's not just a fuzzy end. 675 00:35:05,570 --> 00:35:07,340 It's something that's got a lot of stuff 676 00:35:07,340 --> 00:35:09,770 there that's still base paired. 677 00:35:09,770 --> 00:35:11,330 So the first thing that happens is 678 00:35:11,330 --> 00:35:20,460 that helicase needs to start unzippering 679 00:35:20,460 --> 00:35:25,390 the double-stranded DNA, so you get to a new intermediate. 680 00:35:25,390 --> 00:35:26,760 We're going to draw like this. 681 00:35:41,510 --> 00:35:48,440 And the helicase is going to intercept to basically start 682 00:35:48,440 --> 00:35:53,460 separating those two strands of DNA 683 00:35:53,460 --> 00:35:55,790 in order to do the replication. 684 00:35:55,790 --> 00:35:58,790 But don't forget that these are still base paired, 685 00:35:58,790 --> 00:36:02,910 and these now are single strands. 686 00:36:02,910 --> 00:36:07,170 So I'm going to need something from that menu. 687 00:36:07,170 --> 00:36:08,400 I'm going to need it quickly. 688 00:36:08,400 --> 00:36:10,358 What do you think's the next thing we're really 689 00:36:10,358 --> 00:36:13,140 going to need in order to be able to move forward 690 00:36:13,140 --> 00:36:14,920 in our job? 691 00:36:14,920 --> 00:36:16,350 What do we need from over here? 692 00:36:16,350 --> 00:36:18,940 We're already using the double-stranded DNA. 693 00:36:18,940 --> 00:36:20,940 It's not going to be too difficult to figure out 694 00:36:20,940 --> 00:36:22,020 the NTP's. 695 00:36:22,020 --> 00:36:25,470 We're already using the unzip arrays, the helicase. 696 00:36:25,470 --> 00:36:27,590 What are we going to need? 697 00:36:27,590 --> 00:36:29,890 Yeah. 698 00:36:29,890 --> 00:36:30,940 Primase. 699 00:36:30,940 --> 00:36:32,890 Ah, primase shortly. 700 00:36:32,890 --> 00:36:34,420 But we've really got to do something 701 00:36:34,420 --> 00:36:38,230 to stabilize our unzippered stuff. 702 00:36:38,230 --> 00:36:38,880 Anyone else? 703 00:36:44,430 --> 00:36:48,690 So those two single strands are right near each other, 704 00:36:48,690 --> 00:36:50,610 and they're complementary. 705 00:36:50,610 --> 00:36:53,670 What's going to stop them going straight back together again 706 00:36:53,670 --> 00:36:55,620 and not allowing replication? 707 00:36:55,620 --> 00:36:59,310 So there are proteins that basically 708 00:36:59,310 --> 00:37:07,020 sit on the single-stranded parts of DNA 709 00:37:07,020 --> 00:37:12,270 as are known a single strand binding proteins that stabilize 710 00:37:12,270 --> 00:37:16,260 this transiently made complex long enough for it 711 00:37:16,260 --> 00:37:18,000 to be copied. 712 00:37:18,000 --> 00:37:20,790 So so far, we've used the helicase. 713 00:37:20,790 --> 00:37:25,270 We've used the single strand binding proteins. 714 00:37:25,270 --> 00:37:27,250 And I'm going to draw them there, 715 00:37:27,250 --> 00:37:32,230 still remembering that I have an entire chromosome up here. 716 00:37:35,390 --> 00:37:38,450 Now the heavy lifting has to start fairly soon, 717 00:37:38,450 --> 00:37:40,190 so we need the enzyme that's going 718 00:37:40,190 --> 00:37:44,450 to start making a copy of one of those single strands. 719 00:37:44,450 --> 00:37:47,450 That's going to be a DNA polymerase, which 720 00:37:47,450 --> 00:37:50,210 is right here. 721 00:37:50,210 --> 00:37:53,300 But DNA polymerase is kind of a finicky enzyme 722 00:37:53,300 --> 00:37:56,000 because it doesn't like to just start cold 723 00:37:56,000 --> 00:37:58,070 on the genome, on the single strand, 724 00:37:58,070 --> 00:38:00,410 and start making a copy. 725 00:38:00,410 --> 00:38:04,880 DNA polymerase needs a primer of some kind. 726 00:38:04,880 --> 00:38:08,120 So I'm going to just put that as a note up here. 727 00:38:08,120 --> 00:38:17,450 DNA polymerase needs what's called a primer. 728 00:38:17,450 --> 00:38:18,500 And what is a primer? 729 00:38:18,500 --> 00:38:20,390 A primer is a sequence-- 730 00:38:20,390 --> 00:38:22,490 I think I've got something up here-- 731 00:38:22,490 --> 00:38:26,420 a primer is a sequence that is complementary to a little bit 732 00:38:26,420 --> 00:38:29,630 of DNA so that when DNA polymerase comes along, 733 00:38:29,630 --> 00:38:31,325 it's actually grabbing a double strand 734 00:38:31,325 --> 00:38:34,950 and then filling up the rest of the single strand. 735 00:38:34,950 --> 00:38:38,310 So this would be a typical depiction of a primer. 736 00:38:38,310 --> 00:38:40,710 Here's a strand we want to copy. 737 00:38:40,710 --> 00:38:44,180 But the blue one is a primer strand giving you something 738 00:38:44,180 --> 00:38:46,250 double-stranded to hold onto. 739 00:38:46,250 --> 00:38:49,670 And then in this situation, DNA polymerase 740 00:38:49,670 --> 00:38:53,090 would be happy to fill in the rest of the sequence. 741 00:38:53,090 --> 00:38:56,300 So for the purpose of this discussion to start, 742 00:38:56,300 --> 00:39:01,390 we're just going to provide a primer that 743 00:39:01,390 --> 00:39:05,800 is complementary to the DNA just to give us a go. 744 00:39:05,800 --> 00:39:08,260 We have an alternative in the cell, where 745 00:39:08,260 --> 00:39:11,830 it's to actually use a primase and RNA building 746 00:39:11,830 --> 00:39:13,300 blocks to build a primer. 747 00:39:13,300 --> 00:39:15,340 But let's keep things simple for a minute 748 00:39:15,340 --> 00:39:17,320 and get on with a lot of the major work. 749 00:39:17,320 --> 00:39:19,700 And then we'll come back to that in a second. 750 00:39:19,700 --> 00:39:21,790 So this part is a primer. 751 00:39:25,850 --> 00:39:28,430 And that primer-- if this is the 3 prime end, that's 752 00:39:28,430 --> 00:39:31,940 5 prime 3 prime. 753 00:39:31,940 --> 00:39:35,960 So the primer is complementary to the DNA in that direction. 754 00:39:35,960 --> 00:39:37,880 It's anti-parallel. 755 00:39:37,880 --> 00:39:44,462 And then DNA polymerase can go along and fill in the strand. 756 00:39:44,462 --> 00:39:46,170 And I'm going to draw it dashed, and it's 757 00:39:46,170 --> 00:39:49,940 going to keep on growing. 758 00:39:49,940 --> 00:39:53,240 And what I want you to notice is DNA polymerase 759 00:39:53,240 --> 00:39:59,960 grows from 5 prime to 3 prime. 760 00:39:59,960 --> 00:40:02,990 That is a cardinal rule, the directionality 761 00:40:02,990 --> 00:40:05,450 that we grow the new DNA in. 762 00:40:05,450 --> 00:40:07,850 So basically, looking at this picture, 763 00:40:07,850 --> 00:40:13,100 it's going in this direction because the 3 prime OH is free, 764 00:40:13,100 --> 00:40:14,900 and you're building onto it. 765 00:40:14,900 --> 00:40:17,720 So you use a primer first, and then you 766 00:40:17,720 --> 00:40:34,670 use DNA polymerase plus deoxy nucleoside triphosphates. 767 00:40:34,670 --> 00:40:38,360 So all those AT CG building blocks. 768 00:40:38,360 --> 00:40:40,970 So we've been using these. 769 00:40:40,970 --> 00:40:44,090 We've used the single-stranded binding proteins. 770 00:40:44,090 --> 00:40:48,140 We've used a primer, but there are an alternative. 771 00:40:48,140 --> 00:40:50,030 I'll get to that in just a second. 772 00:40:50,030 --> 00:40:52,940 But we now have a complex where I'm 773 00:40:52,940 --> 00:40:55,580 going to briefly divert you to tell you about, 774 00:40:55,580 --> 00:40:58,760 in the cell, what else you could use as a primer. 775 00:40:58,760 --> 00:41:00,260 And then we're going to have to deal 776 00:41:00,260 --> 00:41:02,280 with copying this other strand. 777 00:41:02,280 --> 00:41:04,670 But this first strand that is made 778 00:41:04,670 --> 00:41:07,130 is called the leading strand. 779 00:41:15,030 --> 00:41:16,200 It's the one that's easy. 780 00:41:16,200 --> 00:41:18,240 I peel apart the DNA. 781 00:41:18,240 --> 00:41:20,910 I have a primer there, and I can just build. 782 00:41:20,910 --> 00:41:22,440 Goes really smoothly. 783 00:41:22,440 --> 00:41:24,160 I'm building in the right direction. 784 00:41:24,160 --> 00:41:26,160 If you look over on the other side of the fence, 785 00:41:26,160 --> 00:41:29,040 you've got a problem because I can't build 786 00:41:29,040 --> 00:41:31,740 on this side from here, right? 787 00:41:31,740 --> 00:41:33,164 Why not? 788 00:41:33,164 --> 00:41:34,192 AUDIENCE: [INAUDIBLE] 789 00:41:34,192 --> 00:41:35,150 BARBARA IMPERIALI: Yes. 790 00:41:35,150 --> 00:41:37,050 I'd be doing it the wrong direction. 791 00:41:37,050 --> 00:41:39,000 I would be the total mess, frankly. 792 00:41:39,000 --> 00:41:41,112 There would be a crisis in the cell. 793 00:41:41,112 --> 00:41:42,820 So we're going to have to deal with that. 794 00:41:42,820 --> 00:41:45,480 So let me just get you out of the primer, just deal 795 00:41:45,480 --> 00:41:47,010 with this primer issue. 796 00:41:47,010 --> 00:41:48,900 In the cell, it's not like we can micro 797 00:41:48,900 --> 00:41:52,380 inject all these little primers to get our DNA synthesized. 798 00:41:52,380 --> 00:42:04,480 So RNA polymerase, believe it or not, doesn't need a primer. 799 00:42:04,480 --> 00:42:09,330 So you can build little bits of primer with RNA. 800 00:42:09,330 --> 00:42:11,853 You don't build very much, just build short segments. 801 00:42:11,853 --> 00:42:13,770 And then once you've got sort of enough there, 802 00:42:13,770 --> 00:42:16,560 DNA polymerase can start polymerizing. 803 00:42:16,560 --> 00:42:23,190 So you use RNA polymerase and nucleoside triphosphates, not 804 00:42:23,190 --> 00:42:25,230 the deoxy ones. 805 00:42:25,230 --> 00:42:27,840 So later on, we've got this piece here 806 00:42:27,840 --> 00:42:31,500 that might be made deoxy RNA, which is why, 807 00:42:31,500 --> 00:42:33,880 coming over here to my menu-- 808 00:42:33,880 --> 00:42:34,520 do I have it? 809 00:42:34,520 --> 00:42:35,280 Yes. 810 00:42:35,280 --> 00:42:39,930 We need an RNA primase, and we need 811 00:42:39,930 --> 00:42:45,720 RNA's to chop them out so that DNA polymerase can come back 812 00:42:45,720 --> 00:42:48,690 because now there is double-stranded material 813 00:42:48,690 --> 00:42:50,740 and fill in the gap. 814 00:42:50,740 --> 00:42:54,870 And then we need one more enzyme to stitch together the gaps. 815 00:42:54,870 --> 00:42:56,280 And that's the ligase. 816 00:42:56,280 --> 00:42:58,050 The logic of this stuff is great. 817 00:42:58,050 --> 00:43:00,990 I think it's just really amazing because all these moving 818 00:43:00,990 --> 00:43:04,200 parts have evolved to make all these functions fall 819 00:43:04,200 --> 00:43:05,320 into place. 820 00:43:05,320 --> 00:43:08,640 Now we need to talk about this pesky other strand. 821 00:43:08,640 --> 00:43:11,730 So we have a problem here because we've 822 00:43:11,730 --> 00:43:13,800 got the wrong directionality. 823 00:43:13,800 --> 00:43:18,550 So what happens in nature is as soon as there's 824 00:43:18,550 --> 00:43:22,450 enough of a stretch, a primer is put in place, 825 00:43:22,450 --> 00:43:25,190 growing the DNA in the right direction. 826 00:43:25,190 --> 00:43:30,880 That's from where the helicase is unzipping. 827 00:43:30,880 --> 00:43:33,160 So we put in another primer. 828 00:43:33,160 --> 00:43:36,960 And then DNA polymerase-- 829 00:43:36,960 --> 00:43:38,900 that's the white one-- 830 00:43:38,900 --> 00:43:42,680 can build its complementary strand, 831 00:43:42,680 --> 00:43:46,730 building in the appropriate direction to be consistent. 832 00:43:46,730 --> 00:43:50,540 So you basically fill in a chunk and then 833 00:43:50,540 --> 00:43:53,540 you have to wait awhile till more is unzippered, 834 00:43:53,540 --> 00:43:56,880 put in another primer, and then grow the DNA. 835 00:43:56,880 --> 00:44:01,550 So in that other piece you're making little segments of DNA 836 00:44:01,550 --> 00:44:04,280 intervened by RNA primers. 837 00:44:04,280 --> 00:44:07,190 Later on, those small primers are going to be chewed up 838 00:44:07,190 --> 00:44:08,840 by RNA's. 839 00:44:08,840 --> 00:44:11,510 DNA polymerase is going to fill them in, 840 00:44:11,510 --> 00:44:13,740 and ligase is going to join them. 841 00:44:13,740 --> 00:44:15,000 Does that make sense? 842 00:44:15,000 --> 00:44:16,370 It's a lot to grasp. 843 00:44:16,370 --> 00:44:19,400 And this other side is called what's 844 00:44:19,400 --> 00:44:23,740 known as the lagging strand. 845 00:44:26,630 --> 00:44:29,360 And there are pieces of short DNA 846 00:44:29,360 --> 00:44:33,980 that are transiently made that have their own name. 847 00:44:33,980 --> 00:44:35,810 You can remember it or not. 848 00:44:35,810 --> 00:44:37,760 I just feel I'm not being complete if I 849 00:44:37,760 --> 00:44:40,090 don't mention their name. 850 00:44:40,090 --> 00:44:47,150 They're called Okazaki fragments after the guy 851 00:44:47,150 --> 00:44:48,740 who discovered them. 852 00:44:48,740 --> 00:44:52,130 And they're longer in bacteria than in humans. 853 00:44:52,130 --> 00:44:56,350 So Okazaki fragments are the transient short stretches 854 00:44:56,350 --> 00:44:59,780 of DNA that are made in the lagging strand, 855 00:44:59,780 --> 00:45:01,500 and then they get zipped together. 856 00:45:01,500 --> 00:45:04,835 So that's sort of the story for replicating the DNA. 857 00:45:04,835 --> 00:45:08,330 Let me make sure I've described everything to you. 858 00:45:08,330 --> 00:45:10,000 Leading strand, lagging strand. 859 00:45:10,000 --> 00:45:13,500 Oh, can't believe I almost forgot this. 860 00:45:13,500 --> 00:45:14,000 All right. 861 00:45:14,000 --> 00:45:15,660 Now we got a problem. 862 00:45:15,660 --> 00:45:18,590 So we've come all this way. 863 00:45:18,590 --> 00:45:19,790 We've used all the pieces. 864 00:45:19,790 --> 00:45:23,600 We haven't used one of the pieces. 865 00:45:23,600 --> 00:45:26,120 So we're in a small bit of trouble 866 00:45:26,120 --> 00:45:32,290 because what's going to happen when you're, for example, 867 00:45:32,290 --> 00:45:35,690 trying to peel apart your DNA? 868 00:45:35,690 --> 00:45:39,180 What trouble are you going to run into? 869 00:45:39,180 --> 00:45:41,930 Who wants to come up here and get involved in my demo? 870 00:45:41,930 --> 00:45:45,540 And you've got to be aware that you're going to be on screen. 871 00:45:45,540 --> 00:45:46,620 You and you up there. 872 00:45:46,620 --> 00:45:48,000 You've got your hand up first. 873 00:45:48,000 --> 00:45:50,270 OK. 874 00:45:50,270 --> 00:45:52,370 I need someone who's going to hold onto this as 875 00:45:52,370 --> 00:45:53,750 if their life depends on it. 876 00:45:53,750 --> 00:45:55,850 You can't let the yellow ones slip out. 877 00:45:55,850 --> 00:45:57,820 So you are the rest of the chromosome, 878 00:45:57,820 --> 00:45:59,960 so make sure that you really hold-- and you 879 00:45:59,960 --> 00:46:01,190 don't let things come apart. 880 00:46:01,190 --> 00:46:03,980 Now you are helicase, OK? 881 00:46:03,980 --> 00:46:04,480 OK. 882 00:46:04,480 --> 00:46:05,120 So come on. 883 00:46:05,120 --> 00:46:09,080 Start helicasing and really pull as hard as you can. 884 00:46:09,080 --> 00:46:11,230 He's going to hold it. 885 00:46:11,230 --> 00:46:13,240 No, I don't want you to unwind it because we're 886 00:46:13,240 --> 00:46:14,490 in the middle of a chromosome. 887 00:46:14,490 --> 00:46:16,960 So you've literally got to do what helicase does. 888 00:46:16,960 --> 00:46:17,950 Yeah, keep on pulling. 889 00:46:17,950 --> 00:46:20,500 Come on, you can do it. 890 00:46:20,500 --> 00:46:21,670 Can you go much further? 891 00:46:21,670 --> 00:46:23,260 I mean, your arms aren't any longer. 892 00:46:23,260 --> 00:46:24,910 But it's almost impossible, right? 893 00:46:24,910 --> 00:46:27,940 We get to a stage where we need help. 894 00:46:27,940 --> 00:46:31,910 The help we have coming is from topoisomerase. 895 00:46:31,910 --> 00:46:36,720 Topoisomerase does something-- so walk just one step this way. 896 00:46:36,720 --> 00:46:37,220 Yeah. 897 00:46:37,220 --> 00:46:42,760 Topoisomerase some comes along and it cuts the DNA. 898 00:46:42,760 --> 00:46:46,930 It holds the pieces in its hands, if you will, 899 00:46:46,930 --> 00:46:49,450 lets the super coiling relax, and then it 900 00:46:49,450 --> 00:46:51,490 joins them back again. 901 00:46:51,490 --> 00:46:53,740 So topoisomerase-- thank you very much, guys. 902 00:46:53,740 --> 00:46:54,400 That's it. 903 00:46:54,400 --> 00:46:56,800 Chromosome, helicase, thank you. 904 00:46:56,800 --> 00:46:58,960 We really have some tightly wound DNA here. 905 00:46:58,960 --> 00:47:03,550 So topoisomerase is the get out of jail free card because it 906 00:47:03,550 --> 00:47:08,410 allows you to deal with all of this tension you got in place 907 00:47:08,410 --> 00:47:10,420 that you cannot unravel. 908 00:47:10,420 --> 00:47:13,510 So you need someone who is literally going to cut, hold, 909 00:47:13,510 --> 00:47:16,080 let the thing relax, rejoin it. 910 00:47:16,080 --> 00:47:18,610 And there are some topoisomerases 911 00:47:18,610 --> 00:47:20,560 called DNA gyrases. 912 00:47:20,560 --> 00:47:23,590 But the really cool thing about topoisomerase 913 00:47:23,590 --> 00:47:26,990 is that it's a wonderful drug target 914 00:47:26,990 --> 00:47:29,860 in both mammalian and bacterial cells 915 00:47:29,860 --> 00:47:34,550 because they're quite different in the two types of organisms. 916 00:47:34,550 --> 00:47:38,350 So in bacteria the antibiotic ciprofloxacin 917 00:47:38,350 --> 00:47:40,930 is a topoisomerase inhibitor that 918 00:47:40,930 --> 00:47:44,890 actually stops your bacteria dividing because if topo 919 00:47:44,890 --> 00:47:46,930 doesn't work, you can't go on. 920 00:47:46,930 --> 00:47:49,760 And in human biology and cancer biology, 921 00:47:49,760 --> 00:47:51,700 there are also topoisomerases-- 922 00:47:51,700 --> 00:47:53,410 there's one called [INAUDIBLE] -- 923 00:47:53,410 --> 00:47:58,030 that does the same thing in the eukaryotic topoisomerase 924 00:47:58,030 --> 00:48:03,820 to stop cancer cells dividing rapidly so that those cannot go 925 00:48:03,820 --> 00:48:06,310 on and make larger tumors. 926 00:48:06,310 --> 00:48:07,720 So I want to show you something. 927 00:48:07,720 --> 00:48:11,560 And I am actually certain that I have time. 928 00:48:11,560 --> 00:48:12,910 Come on. 929 00:48:12,910 --> 00:48:14,030 Now I'm going to show you. 930 00:48:14,030 --> 00:48:14,697 [VIDEO PLAYBACK] 931 00:48:14,697 --> 00:48:16,980 - In this animation, we'll see the remarkable way 932 00:48:16,980 --> 00:48:20,140 our DNA is tightly packed up so that six 933 00:48:20,140 --> 00:48:23,860 feet of this long molecule fits into the microscopic nucleus 934 00:48:23,860 --> 00:48:24,790 of every cell. 935 00:48:28,450 --> 00:48:31,900 The process starts when DNA is wrapped around special protein 936 00:48:31,900 --> 00:48:34,440 molecules called histones. 937 00:48:34,440 --> 00:48:38,050 The combined loop of DNA and protein is called a nucleosome. 938 00:48:42,500 --> 00:48:46,510 Next, the nucleosomes are packaged into a thread. 939 00:48:46,510 --> 00:48:49,070 The end result is a fiber known as chromatin. 940 00:48:55,860 --> 00:49:17,380 This fiber is then looped and coiled yet again, 941 00:49:17,380 --> 00:49:19,540 leading finally to the familiar shapes known 942 00:49:19,540 --> 00:49:23,180 as chromosomes, which can be seen in the nucleus of dividing 943 00:49:23,180 --> 00:49:23,680 cells. 944 00:49:27,748 --> 00:49:32,130 BARBARA IMPERIALI: Now I'm going to take you forward to DNA, 945 00:49:32,130 --> 00:49:33,990 what we just talked about here. 946 00:49:37,290 --> 00:49:40,680 - Using computer animation based on molecular research, 947 00:49:40,680 --> 00:49:43,320 we are now able to see how DNA is actually 948 00:49:43,320 --> 00:49:44,490 copied in living cells. 949 00:49:47,170 --> 00:49:48,880 You are looking at an assembly line 950 00:49:48,880 --> 00:49:52,600 of amazing miniature biochemical machines that are pulling apart 951 00:49:52,600 --> 00:49:55,090 the DNA double helix and cranking out 952 00:49:55,090 --> 00:49:56,330 a copy of each strand. 953 00:49:59,700 --> 00:50:02,370 The DNA to be copied enters the production line 954 00:50:02,370 --> 00:50:04,770 from bottom left. 955 00:50:04,770 --> 00:50:09,210 The whirling blue molecular machine is called helicase. 956 00:50:09,210 --> 00:50:12,360 It spins the DNA as fast as a jet engine 957 00:50:12,360 --> 00:50:17,220 as it unwinds the double helix into two strands. 958 00:50:17,220 --> 00:50:19,890 One strand is copied continuously 959 00:50:19,890 --> 00:50:22,000 and can be seen spooling off to the right. 960 00:50:24,870 --> 00:50:27,360 Things are not so simple for the other strand 961 00:50:27,360 --> 00:50:30,780 because it must be copied backwards. 962 00:50:30,780 --> 00:50:32,670 It is drawn out repeatedly in loops 963 00:50:32,670 --> 00:50:36,330 and copied one section at a time. 964 00:50:36,330 --> 00:50:39,210 The end result is two new DNA molecules. 965 00:50:44,367 --> 00:50:44,950 [END PLAYBACK] 966 00:50:44,950 --> 00:50:46,158 BARBARA IMPERIALI: All right. 967 00:50:46,158 --> 00:50:47,420 OK. 968 00:50:47,420 --> 00:50:50,450 That's what you saw today, replication, 969 00:50:50,450 --> 00:50:53,990 a lot of the mechanics, a lot of the moving parts. 970 00:50:53,990 --> 00:50:56,480 There's this tricky stuff with primers. 971 00:50:56,480 --> 00:50:57,960 You'll get used to that. 972 00:50:57,960 --> 00:51:00,110 But this is really the quintessential set 973 00:51:00,110 --> 00:51:02,840 of pieces to replicate DNA. 974 00:51:02,840 --> 00:51:04,700 It is an amazing process. 975 00:51:04,700 --> 00:51:06,140 Look at the speed. 976 00:51:06,140 --> 00:51:08,270 1,000 base pairs a second. 977 00:51:08,270 --> 00:51:10,640 Can you even believe it? 978 00:51:10,640 --> 00:51:16,640 An entire circular chromosome copied in 20 minutes. 979 00:51:16,640 --> 00:51:18,590 So these are really things to think about 980 00:51:18,590 --> 00:51:22,160 because they are so impressive that it's a delight to actually 981 00:51:22,160 --> 00:51:23,610 be able to teach them to you. 982 00:51:23,610 --> 00:51:24,110 OK. 983 00:51:24,110 --> 00:51:26,230 I'm done for today.