1 00:00:06,370 --> 00:00:12,480 ERIC LANDER: And so the issue became how does DNA 2 00:00:12,480 --> 00:00:13,875 replication work. 3 00:00:17,370 --> 00:00:18,650 And so I'm about to go into it. 4 00:00:18,650 --> 00:00:25,130 Now, I'm going to note we're going to be starting this DNA 5 00:00:25,130 --> 00:00:35,790 goes to RNA, goes to protein, and DNA goes to itself. 6 00:00:35,790 --> 00:00:38,100 DNA is replicated. 7 00:00:38,100 --> 00:00:39,450 It makes RNA. 8 00:00:39,450 --> 00:00:41,710 The RNA is used to make protein. 9 00:00:41,710 --> 00:00:42,780 This will be what we'll be talking 10 00:00:42,780 --> 00:00:44,700 about today and tomorrow. 11 00:00:44,700 --> 00:00:47,640 So the first step of that is, how does DNA give 12 00:00:47,640 --> 00:00:50,370 rise to more DNA? 13 00:00:50,370 --> 00:00:53,330 Well, how do you find an enzyme? 14 00:00:53,330 --> 00:00:54,580 How do you do biochemistry? 15 00:00:56,950 --> 00:00:57,540 What do you do? 16 00:00:57,540 --> 00:00:58,890 AUDIENCE: Assays. 17 00:00:58,890 --> 00:00:59,350 ERIC LANDER: Assay. 18 00:00:59,350 --> 00:01:00,730 So you've got to grind up the cell. 19 00:01:00,730 --> 00:01:02,730 I got to choose a cell in which I'm likely to find an 20 00:01:02,730 --> 00:01:05,830 enzyme, grind it up, break it up into different fractions, 21 00:01:05,830 --> 00:01:07,200 and test each fraction. 22 00:01:07,200 --> 00:01:08,930 That's all biochemists do, right? 23 00:01:08,930 --> 00:01:13,950 So what cell might have the enzyme we're looking for? 24 00:01:13,950 --> 00:01:15,675 What cells might be able to copy DNA? 25 00:01:18,642 --> 00:01:20,400 How about all cells? 26 00:01:20,400 --> 00:01:21,570 So let's use a simple cell. 27 00:01:21,570 --> 00:01:24,180 What's a simple cell? 28 00:01:24,180 --> 00:01:25,330 Let's use bacteria. 29 00:01:25,330 --> 00:01:27,000 So we'll take some bacteria, we'll grow it up, 30 00:01:27,000 --> 00:01:27,920 we'll grind it up. 31 00:01:27,920 --> 00:01:30,130 We'll fractionate it into different fractions, and we'll 32 00:01:30,130 --> 00:01:33,810 see if one of those fractions has the ability to copy DNA. 33 00:01:33,810 --> 00:01:35,150 If we're going to run an assay, we have 34 00:01:35,150 --> 00:01:36,880 to give it a substrate. 35 00:01:36,880 --> 00:01:40,610 What substrate would you like to give it? 36 00:01:40,610 --> 00:01:41,760 What do you think it needs? 37 00:01:41,760 --> 00:01:43,010 AUDIENCE: [INAUDIBLE]. 38 00:01:45,450 --> 00:01:47,380 ERIC LANDER: It better have some free nucleotides 39 00:01:47,380 --> 00:01:48,980 otherwise, how are we going to make DNA. 40 00:01:48,980 --> 00:01:51,010 What else? 41 00:01:51,010 --> 00:01:53,490 Are you going to ask it to make DNA all by itself? 42 00:01:53,490 --> 00:01:56,110 We want something that can copy one of the strands of a 43 00:01:56,110 --> 00:01:57,310 double helix. 44 00:01:57,310 --> 00:01:58,140 So what should we give it? 45 00:01:58,140 --> 00:02:00,240 AUDIENCE: [INAUDIBLE]. 46 00:02:00,240 --> 00:02:01,150 ERIC LANDER: Sorry? 47 00:02:01,150 --> 00:02:02,020 AUDIENCE: Half a helix. 48 00:02:02,020 --> 00:02:02,850 ERIC LANDER: Half a helix. 49 00:02:02,850 --> 00:02:06,480 A strand of DNA, the strand to be used as a template. 50 00:02:06,480 --> 00:02:07,965 So let's give it a template strand. 51 00:02:11,880 --> 00:02:14,860 So we'll take a template strand of DNA. 52 00:02:14,860 --> 00:02:16,390 There's my template of DNA. 53 00:02:19,600 --> 00:02:22,810 Let's actually give it a little 54 00:02:22,810 --> 00:02:24,980 sequence actually, here. 55 00:02:24,980 --> 00:02:34,330 Let's say A phosphate, T phosphate, G phosphate, C 56 00:02:34,330 --> 00:02:41,480 phosphate, A phosphate, T phosphate, T phosphate, A 57 00:02:41,480 --> 00:02:46,460 phosphate, G phosphate, G phosphate. 58 00:02:46,460 --> 00:02:48,810 I'm going to not write the phosphates too much longer, 59 00:02:48,810 --> 00:02:51,760 guys, but anyway C phosphate, C phosphate, T 60 00:02:51,760 --> 00:02:55,060 phosphate, like that. 61 00:02:55,060 --> 00:02:57,410 Pretty soon, in fact, almost immediately, I'm going to 62 00:02:57,410 --> 00:02:59,820 start dropping the phosphates in here. 63 00:02:59,820 --> 00:03:01,930 But that's the way it goes. 64 00:03:01,930 --> 00:03:04,610 All right. 65 00:03:04,610 --> 00:03:05,860 That's a template. 66 00:03:08,640 --> 00:03:14,845 We need floating around in the solution some trinucleotides. 67 00:03:25,960 --> 00:03:33,230 We have some nucleotides floating around. 68 00:03:36,160 --> 00:03:39,330 And now will this enzyme work? 69 00:03:39,330 --> 00:03:41,540 We would try different fractions and see if it's able 70 00:03:41,540 --> 00:03:45,940 to just install the right letters in the right place. 71 00:03:45,940 --> 00:03:50,620 Now, it turned out it needed one more thing, and the person 72 00:03:50,620 --> 00:03:53,370 who discovered this, Arthur Kornberg, thought of it. 73 00:03:53,370 --> 00:03:54,760 It needed a head start. 74 00:03:54,760 --> 00:03:56,010 It needed a primer. 75 00:03:59,450 --> 00:04:05,690 So the primer goes let's say, phosphate T, phosphate A, 76 00:04:05,690 --> 00:04:16,670 phosphate C, phosphate G, phosphate T, phosphate A, 77 00:04:16,670 --> 00:04:18,399 let's say like that. 78 00:04:18,399 --> 00:04:23,950 So this is the five prime end of DNA. 79 00:04:23,950 --> 00:04:26,490 Remember the phosphate is hanging off the five prime 80 00:04:26,490 --> 00:04:27,940 carbon, right? 81 00:04:27,940 --> 00:04:29,400 What's look at the other end. 82 00:04:29,400 --> 00:04:34,640 The other end ends in the hydroxyl on the three prime 83 00:04:34,640 --> 00:04:37,790 end of the ribose. 84 00:04:37,790 --> 00:04:42,010 Since this is anti-parallel, this strand is going five 85 00:04:42,010 --> 00:04:48,090 prime phosphate to three prime hydroxyl. 86 00:04:48,090 --> 00:04:50,540 You're going to need to know five prime and three prime. 87 00:04:50,540 --> 00:04:52,470 So I'm doing this so you get used to five 88 00:04:52,470 --> 00:04:53,880 prime and three prime. 89 00:04:53,880 --> 00:04:54,580 There you go. 90 00:04:54,580 --> 00:04:57,460 If you're handed a primer to get a head start, and you're 91 00:04:57,460 --> 00:05:00,460 handed a template, and you hand it some nucleotides, you 92 00:05:00,460 --> 00:05:03,800 then assay different fractions exactly as you suggested and 93 00:05:03,800 --> 00:05:09,300 we see is one of them capable of extending this strand by 94 00:05:09,300 --> 00:05:12,640 putting in an A, putting in a T, putting in a C, putting in 95 00:05:12,640 --> 00:05:17,320 a C, putting in a C, putting in a G. That's the assay. 96 00:05:17,320 --> 00:05:29,450 And Arthur Kornberg discovered an enzyme that could do this. 97 00:05:29,450 --> 00:05:31,000 And the biochemists went nuts. 98 00:05:31,000 --> 00:05:31,940 They thought, wow. 99 00:05:31,940 --> 00:05:33,520 This is so cool. 100 00:05:33,520 --> 00:05:36,770 Kornberg is able to discover an enzyme that 101 00:05:36,770 --> 00:05:39,220 can accomplish this. 102 00:05:39,220 --> 00:05:43,470 The enzyme polymerizes DNA. 103 00:05:43,470 --> 00:05:46,754 Coincidentally, what is the enzyme called? 104 00:05:46,754 --> 00:05:47,660 AUDIENCE: DNA polymerase. 105 00:05:47,660 --> 00:05:49,320 ERIC LANDER: DNA polymerase. 106 00:05:49,320 --> 00:05:51,130 Accidentally, has a nice name. 107 00:05:51,130 --> 00:05:52,310 Good. 108 00:05:52,310 --> 00:05:54,050 DNA polymerase. 109 00:05:58,590 --> 00:06:01,190 Excellent. 110 00:06:01,190 --> 00:06:03,640 Now, notice what it does. 111 00:06:03,640 --> 00:06:08,130 It takes this triphosphate, puts it in here, and it joins 112 00:06:08,130 --> 00:06:10,140 it into a sugar phosphate chain. 113 00:06:10,140 --> 00:06:12,205 Where does it get the energy for that synthesis? 114 00:06:14,780 --> 00:06:17,600 Hydrolysis of the triphosphates, right? 115 00:06:17,600 --> 00:06:19,500 It's the hydrolysis of the triphosphate. 116 00:06:19,500 --> 00:06:21,800 That's the energy. 117 00:06:21,800 --> 00:06:26,030 What direction is the synthesis proceeding? 118 00:06:26,030 --> 00:06:31,120 Starts here at the five prime end, and it moves adding on to 119 00:06:31,120 --> 00:06:33,400 the three prime end. 120 00:06:33,400 --> 00:06:40,880 So it's five prime to three prime direction. 121 00:06:40,880 --> 00:06:42,540 That's the direction it moves. 122 00:06:42,540 --> 00:06:44,720 It adds to the three prime end. 123 00:06:44,720 --> 00:06:46,370 It adds to the free nucleotides to 124 00:06:46,370 --> 00:06:47,620 the three prime end. 125 00:06:50,350 --> 00:06:52,025 Why not do it the other way? 126 00:06:52,025 --> 00:06:55,140 AUDIENCE: [INAUDIBLE]. 127 00:06:55,140 --> 00:06:56,170 ERIC LANDER: Sorry? 128 00:06:56,170 --> 00:06:57,370 AUDIENCE: Phosphates. 129 00:06:57,370 --> 00:06:58,286 ERIC LANDER: Can't hear you. 130 00:06:58,286 --> 00:06:58,700 Shout loud. 131 00:06:58,700 --> 00:06:59,586 AUDIENCE: Phosphates. 132 00:06:59,586 --> 00:07:02,000 ERIC LANDER: Phosphates, yes. 133 00:07:02,000 --> 00:07:05,075 You see, suppose we were going the other way. 134 00:07:08,850 --> 00:07:11,540 Suppose the primer was this way. 135 00:07:11,540 --> 00:07:15,600 Where would as we added each base, the triphosphate would 136 00:07:15,600 --> 00:07:18,590 be on the strands, right? 137 00:07:18,590 --> 00:07:24,150 And we'd be adding to the three prime end here. 138 00:07:24,150 --> 00:07:28,320 That means the energy supplied by the triphosphate would be 139 00:07:28,320 --> 00:07:33,240 on the growing strands rather than in the free nucleotides. 140 00:07:33,240 --> 00:07:37,160 Why would it be a terrible idea to put your energy source 141 00:07:37,160 --> 00:07:39,729 on the growing strand? 142 00:07:39,729 --> 00:07:42,094 MIKE: [INAUDIBLE]. 143 00:07:42,094 --> 00:07:44,280 ERIC LANDER: Well Mike, you know, those triphosphate bonds 144 00:07:44,280 --> 00:07:45,040 are pretty unstable. 145 00:07:45,040 --> 00:07:47,320 They hydrolyzed by themselves at some frequency. 146 00:07:47,320 --> 00:07:49,550 If you're a free nucleotide and the triphosphate 147 00:07:49,550 --> 00:07:52,010 hydrolyzes, big deal. 148 00:07:52,010 --> 00:07:55,900 That free nucleotide floating around loses its triphosphate. 149 00:07:55,900 --> 00:07:57,960 But what if I'm the growing strand, and I lose my 150 00:07:57,960 --> 00:07:59,115 triphosphate? 151 00:07:59,115 --> 00:07:59,720 AUDIENCE: [LAUGHS] 152 00:07:59,720 --> 00:08:00,480 ERIC LANDER: Exactly. 153 00:08:00,480 --> 00:08:01,540 AUDIENCE: There goes your chain. 154 00:08:01,540 --> 00:08:03,470 ERIC LANDER: There goes my chain. 155 00:08:03,470 --> 00:08:05,530 So you know, life's not stupid. 156 00:08:05,530 --> 00:08:06,930 It doesn't do it that way. 157 00:08:06,930 --> 00:08:07,870 It does it this way. 158 00:08:07,870 --> 00:08:10,050 No one has ever found a polymerase that goes this way. 159 00:08:10,050 --> 00:08:13,230 They find them all going that way for just that reason. 160 00:08:13,230 --> 00:08:14,660 Exactly. 161 00:08:14,660 --> 00:08:16,100 Bingo. 162 00:08:16,100 --> 00:08:19,210 That was why life evolved it that way, because you want 163 00:08:19,210 --> 00:08:24,190 your triphosphates, those hydrolyzable triphosphates to 164 00:08:24,190 --> 00:08:27,320 be floating around freely rather than investing. 165 00:08:27,320 --> 00:08:28,270 Now just think about that. 166 00:08:28,270 --> 00:08:29,140 It's a kind of cool thing. 167 00:08:29,140 --> 00:08:29,670 It doesn't matter. 168 00:08:29,670 --> 00:08:30,910 Your book doesn't talk about it. 169 00:08:30,910 --> 00:08:33,080 But to me, it helps me remember which way it's going 170 00:08:33,080 --> 00:08:35,370 and how it is, and it's kind of interesting. 171 00:08:35,370 --> 00:08:36,110 Any way. 172 00:08:36,110 --> 00:08:36,710 All right. 173 00:08:36,710 --> 00:08:42,440 So Kornberg wins the Nobel Prize for this. 174 00:08:42,440 --> 00:08:42,870 Good stuff. 175 00:08:42,870 --> 00:08:47,920 It's very deserved, but you know, there's some questions. 176 00:08:47,920 --> 00:08:52,340 Where does the primer come from in life? 177 00:08:52,340 --> 00:08:57,270 See, Kornberg gave this test tube a primer. 178 00:08:57,270 --> 00:09:00,520 But suppose I'm replicating some DNA. 179 00:09:00,520 --> 00:09:06,770 So let's suppose I have a double strand of DNA, and I'm 180 00:09:06,770 --> 00:09:13,460 just going to open it up here, five prime to three prime, 181 00:09:13,460 --> 00:09:18,280 five prime to three prime. 182 00:09:18,280 --> 00:09:20,769 I need to get like a primer here. 183 00:09:25,080 --> 00:09:28,830 Then the primer can be extended by polymerase. 184 00:09:28,830 --> 00:09:32,560 Well, where's the primer come from? 185 00:09:32,560 --> 00:09:35,850 It turns out there is an enzyme specially devoted to 186 00:09:35,850 --> 00:09:37,440 making those primers. 187 00:09:37,440 --> 00:09:41,080 Kornberg didn't know it, but there's an enzyme. 188 00:09:41,080 --> 00:09:46,420 And by coincidence, it is called primase. 189 00:09:46,420 --> 00:09:48,240 Exactly. 190 00:09:48,240 --> 00:09:50,290 Primase makes the primer. 191 00:09:50,290 --> 00:09:56,025 So you need a primer here, and the primer is made by primase. 192 00:10:00,960 --> 00:10:07,810 Once primase makes a primer, polymerase can chug along and 193 00:10:07,810 --> 00:10:10,340 do it just fine. 194 00:10:10,340 --> 00:10:11,860 Let's check out the other strand. 195 00:10:14,370 --> 00:10:18,390 Primer here, polymerase chugs along. 196 00:10:21,790 --> 00:10:25,210 But now as this double helix opens up, what 197 00:10:25,210 --> 00:10:26,460 happens over here? 198 00:10:31,380 --> 00:10:34,150 The synthesis going this way. 199 00:10:34,150 --> 00:10:35,670 So what do I have to do here? 200 00:10:35,670 --> 00:10:37,318 AUDIENCE: [INAUDIBLE]. 201 00:10:37,318 --> 00:10:39,232 ERIC LANDER: Another primer. 202 00:10:39,232 --> 00:10:40,482 Need another primer. 203 00:10:42,810 --> 00:10:44,510 Then as it opens up more, what do I need? 204 00:10:44,510 --> 00:10:45,530 AUDIENCE: Another primer. 205 00:10:45,530 --> 00:10:46,780 ERIC LANDER: Another primer. 206 00:10:49,410 --> 00:10:53,100 So the two strands are experiencing very different 207 00:10:53,100 --> 00:10:54,450 kind of replication. 208 00:10:54,450 --> 00:10:57,420 In one place, one primer in the five prime to three prime 209 00:10:57,420 --> 00:10:59,840 direction is enough to keep going. 210 00:10:59,840 --> 00:11:02,830 In the other strand, as it keeps opening up, you gotta 211 00:11:02,830 --> 00:11:04,510 keep making primers. 212 00:11:04,510 --> 00:11:06,550 You have all these little fragments there. 213 00:11:09,910 --> 00:11:16,520 Now, those little fragments were discovered by Okazaki, 214 00:11:16,520 --> 00:11:20,960 and they are called Okazaki fragments. 215 00:11:20,960 --> 00:11:23,260 Again, I just mention these things. 216 00:11:23,260 --> 00:11:25,570 They are known to molecular biologists. 217 00:11:25,570 --> 00:11:28,900 But these little guys are Okazaki fragments, and they 218 00:11:28,900 --> 00:11:31,480 tell you that you're on the right track here. 219 00:11:31,480 --> 00:11:33,280 This is indeed how it's working. 220 00:11:33,280 --> 00:11:36,010 You can see those little fragments there. 221 00:11:36,010 --> 00:11:40,260 But now, what's the problem with the Okazaki fragments? 222 00:11:40,260 --> 00:11:41,970 They're not connected, right? 223 00:11:41,970 --> 00:11:44,400 The primase makes a primer. 224 00:11:44,400 --> 00:11:50,010 The polymerase copies the DNA, it bumps into the next primer, 225 00:11:50,010 --> 00:11:52,690 but you've got to connect them. 226 00:11:52,690 --> 00:11:54,420 So that's a problem. 227 00:11:54,420 --> 00:11:57,250 That's a real problem. 228 00:11:57,250 --> 00:11:58,500 I'll redraw that here. 229 00:12:04,140 --> 00:12:05,160 Here was my primer. 230 00:12:05,160 --> 00:12:06,710 I got a new primer over here. 231 00:12:09,450 --> 00:12:12,680 I got a new primer over here. 232 00:12:12,680 --> 00:12:13,380 Right there. 233 00:12:13,380 --> 00:12:14,120 Right there. 234 00:12:14,120 --> 00:12:17,340 They're not contiguous connected. 235 00:12:17,340 --> 00:12:20,820 The word we use for connecting two pieces of DNA, which is a 236 00:12:20,820 --> 00:12:23,700 standard English word not used that often is to ligate two 237 00:12:23,700 --> 00:12:25,060 things together. 238 00:12:25,060 --> 00:12:27,960 Ligature, for example, in music. 239 00:12:27,960 --> 00:12:31,460 You ligate things together. 240 00:12:31,460 --> 00:12:33,650 How do you think the cell deals with ligating these 241 00:12:33,650 --> 00:12:36,230 things together? 242 00:12:36,230 --> 00:12:37,315 An enzyme called-- 243 00:12:37,315 --> 00:12:38,110 AUDIENCE: Ligase. 244 00:12:38,110 --> 00:12:40,390 ERIC LANDER: Exactly. 245 00:12:40,390 --> 00:12:44,340 So ligase does the ligation. 246 00:12:44,340 --> 00:12:48,070 Ligase ligates. 247 00:12:48,070 --> 00:12:51,950 It is so lucky that these words turn out to have 248 00:12:51,950 --> 00:12:53,780 accidentally made sense. 249 00:12:53,780 --> 00:12:56,400 It's really cool. 250 00:12:56,400 --> 00:12:59,950 So ligase ligates. 251 00:12:59,950 --> 00:13:05,000 Now, I'll tell you a factoid, but don't worry 252 00:13:05,000 --> 00:13:05,820 about it too much. 253 00:13:05,820 --> 00:13:10,360 Primase actually doesn't make DNA. 254 00:13:10,360 --> 00:13:11,540 We haven't gotten there yet, but it turns out 255 00:13:11,540 --> 00:13:14,350 primase makes RNA. 256 00:13:14,350 --> 00:13:16,450 Turns out to be easier to start an RNA 257 00:13:16,450 --> 00:13:18,740 than a DNA from scratch. 258 00:13:18,740 --> 00:13:20,710 Cell doesn't like to start DNA from scratch. 259 00:13:20,710 --> 00:13:22,650 It likes to start RNA from scratch as we'll get to a 260 00:13:22,650 --> 00:13:23,870 moment with transcription. 261 00:13:23,870 --> 00:13:26,010 So as a factoid, I'll mention to you that those little 262 00:13:26,010 --> 00:13:29,340 primers are actually RNA primers, and what happens is 263 00:13:29,340 --> 00:13:32,800 they get extended into DNA, and they bump into and kind of 264 00:13:32,800 --> 00:13:36,070 displace the previous RNA, so it's slightly more complicated 265 00:13:36,070 --> 00:13:36,890 than I told you. 266 00:13:36,890 --> 00:13:38,270 You're welcome to forget that. 267 00:13:38,270 --> 00:13:40,290 If you would like to believe that primase is actually 268 00:13:40,290 --> 00:13:43,550 making little segments of DNA, it'll be just fine. 269 00:13:43,550 --> 00:13:45,460 But in fact, it doesn't actually. 270 00:13:45,460 --> 00:13:47,990 It's making little segments of RNA so there's a whole other 271 00:13:47,990 --> 00:13:50,070 machinery that has to deal with that. 272 00:13:50,070 --> 00:13:52,810 But the basic concept five prime to three prime, little 273 00:13:52,810 --> 00:13:56,765 primers, getting extended, getting ligated, that's how 274 00:13:56,765 --> 00:13:57,620 you make your DNA. 275 00:13:57,620 --> 00:13:58,980 And you can check it out, and it works. 276 00:13:58,980 --> 00:14:00,230 All right. 277 00:14:09,500 --> 00:14:17,770 Well, it turns out to even be a little more complicated. 278 00:14:17,770 --> 00:14:20,920 That was how we got the synthesis going, but we also 279 00:14:20,920 --> 00:14:23,030 have a little bit of a topological problem. 280 00:14:31,090 --> 00:14:35,120 This again, says a lot about how people do science. 281 00:14:35,120 --> 00:14:37,300 You gotta just like not worry about certain things. 282 00:14:37,300 --> 00:14:40,390 If Kornberg had said, oh my goodness. 283 00:14:40,390 --> 00:14:43,860 I can't give my test tube a primer, because I don't know 284 00:14:43,860 --> 00:14:46,550 how the cell would make a primer, he wouldn't have made 285 00:14:46,550 --> 00:14:47,120 any progress. 286 00:14:47,120 --> 00:14:49,023 So he throws in the primer and says, the cell 287 00:14:49,023 --> 00:14:50,070 will figure it out. 288 00:14:50,070 --> 00:14:54,400 I'm just giving it a primer, and I'll see what happens. 289 00:14:54,400 --> 00:14:56,820 Now, there's another problem, this topological problem that 290 00:14:56,820 --> 00:14:58,300 also can make your head hurt. 291 00:14:58,300 --> 00:15:01,050 Let me try to explain what the topological problem is. 292 00:15:01,050 --> 00:15:08,735 Suppose I have DNA like that. 293 00:15:08,735 --> 00:15:10,580 Make that a little prettier. 294 00:15:10,580 --> 00:15:12,110 So I have some DNA like that. 295 00:15:21,570 --> 00:15:24,360 And maybe it goes around for a very long distance like a 296 00:15:24,360 --> 00:15:25,800 circle or something like that. 297 00:15:25,800 --> 00:15:30,070 I now want to copy that DNA. 298 00:15:30,070 --> 00:15:33,390 So I have one strand, and I'm copying it. 299 00:15:36,730 --> 00:15:43,650 I have this other strand, and I'm copying it. 300 00:15:43,650 --> 00:15:47,760 And remember, these two strands are wrapped around, 301 00:15:47,760 --> 00:15:51,560 and around, and around, and around each other. 302 00:15:51,560 --> 00:15:53,170 One is going like this. 303 00:15:53,170 --> 00:15:55,050 One is going like that, and there's some wrapped around. 304 00:15:55,050 --> 00:15:59,430 And as I tug them apart to make a new strand, to 305 00:15:59,430 --> 00:16:05,560 synthesize a new strand, those two new double helices are so 306 00:16:05,560 --> 00:16:10,360 totally intertwined with each other. 307 00:16:10,360 --> 00:16:14,720 Every turn that there was in the double helix is now a 308 00:16:14,720 --> 00:16:18,780 twist and turn connecting the two, sort of entangling the 309 00:16:18,780 --> 00:16:20,380 two helices. 310 00:16:20,380 --> 00:16:23,440 So I have the two new double helices 311 00:16:23,440 --> 00:16:26,130 entangled with each other. 312 00:16:26,130 --> 00:16:27,380 Why is that going to be a problem? 313 00:16:31,160 --> 00:16:34,070 I'm going to send these to two daughter cells. 314 00:16:34,070 --> 00:16:37,060 These are the two genomes for the two daughter cells. 315 00:16:37,060 --> 00:16:39,990 In fact in particular, if this thing was a circle, the two 316 00:16:39,990 --> 00:16:44,360 new circles will be totally wrapped around each other with 317 00:16:44,360 --> 00:16:46,650 a gazillion wraps. 318 00:16:46,650 --> 00:16:50,130 No way they're going to two daughter cells. 319 00:16:50,130 --> 00:16:51,560 Now, here is where mathematicians are very 320 00:16:51,560 --> 00:16:55,120 useful, because it is a theorem that if I take two 321 00:16:55,120 --> 00:16:59,020 circles wrapped around each other like that, there is no 322 00:16:59,020 --> 00:17:01,750 topological deformation possible that 323 00:17:01,750 --> 00:17:02,590 can separate them. 324 00:17:02,590 --> 00:17:04,359 It's like these puzzles, you get some strings wrapped 325 00:17:04,359 --> 00:17:06,240 around each other separate them. 326 00:17:06,240 --> 00:17:08,960 It's a theorem that two circles wrapped around each 327 00:17:08,960 --> 00:17:13,839 other like that cannot be separated unless, 328 00:17:13,839 --> 00:17:14,520 of course, you cheat. 329 00:17:14,520 --> 00:17:15,352 What's cheating? 330 00:17:15,352 --> 00:17:16,500 AUDIENCE: You cut it. 331 00:17:16,500 --> 00:17:17,319 ERIC LANDER: You cut it, obviously. 332 00:17:17,319 --> 00:17:18,980 If you cut it, then you can separate it. 333 00:17:18,980 --> 00:17:20,670 But otherwise, it's mathematically impossible to 334 00:17:20,670 --> 00:17:22,430 separate them. 335 00:17:22,430 --> 00:17:25,349 So this could concern people. 336 00:17:25,349 --> 00:17:27,450 How could a cell do this? 337 00:17:27,450 --> 00:17:28,867 So what does the cell do? 338 00:17:28,867 --> 00:17:29,781 AUDIENCE: It cuts it. 339 00:17:29,781 --> 00:17:30,820 ERIC LANDER: It cuts it. 340 00:17:30,820 --> 00:17:32,020 It's got no choice, right? 341 00:17:32,020 --> 00:17:32,990 It's a theorem, right? 342 00:17:32,990 --> 00:17:35,050 Even cells can't violate theorems. 343 00:17:35,050 --> 00:17:38,310 So it cuts it. 344 00:17:38,310 --> 00:17:40,440 The only way to get these things apart is to cut it. 345 00:17:40,440 --> 00:17:43,570 Now, what it does, is it takes those double helices. 346 00:17:43,570 --> 00:17:46,030 I'll represent the double helix as a thicker 347 00:17:46,030 --> 00:17:46,620 kind of thing now. 348 00:17:46,620 --> 00:17:49,480 That was my double helix, this other double helix 349 00:17:49,480 --> 00:17:52,200 wrapped around it. 350 00:17:52,200 --> 00:17:55,150 It's got to cut it. 351 00:17:55,150 --> 00:18:00,840 Now, when I take two DNAs that are wrapped around each other 352 00:18:00,840 --> 00:18:02,720 or two DNAs that are separate, have I done any 353 00:18:02,720 --> 00:18:04,920 chemistry on them? 354 00:18:04,920 --> 00:18:05,160 I'm sorry. 355 00:18:05,160 --> 00:18:07,040 Are they chemically different? 356 00:18:07,040 --> 00:18:09,440 They're chemically the same molecules. 357 00:18:09,440 --> 00:18:13,110 But they're topologically different. 358 00:18:13,110 --> 00:18:15,150 Topologically means wrapped around. 359 00:18:15,150 --> 00:18:17,160 In one case, they were topologically entangled. 360 00:18:17,160 --> 00:18:19,510 In the other case, they're topologically separated from 361 00:18:19,510 --> 00:18:20,280 each other. 362 00:18:20,280 --> 00:18:23,030 So they're still the same chemical bonds, the same 363 00:18:23,030 --> 00:18:29,260 molecules, but when I separate these two double helices now, 364 00:18:29,260 --> 00:18:32,220 the difference between these is that they are what are 365 00:18:32,220 --> 00:18:35,860 called topoisomers. 366 00:18:35,860 --> 00:18:38,620 They are isomers because they're exactly the same 367 00:18:38,620 --> 00:18:39,920 chemical formula. 368 00:18:39,920 --> 00:18:41,940 But they're topoisomers because they 369 00:18:41,940 --> 00:18:43,070 have different topology. 370 00:18:43,070 --> 00:18:46,060 They're not wrapped around each other anymore. 371 00:18:46,060 --> 00:18:48,900 So it turns out there is an enzyme that just gets in there 372 00:18:48,900 --> 00:18:51,040 and makes a double stranded cut in one of the double 373 00:18:51,040 --> 00:18:54,920 helices, grabs the two ends, passes it around the other 374 00:18:54,920 --> 00:18:59,380 side, and ligates them back together, and keeps doing that 375 00:18:59,380 --> 00:19:01,410 until they're disentangled. 376 00:19:01,410 --> 00:19:02,960 Pretty clever. 377 00:19:02,960 --> 00:19:06,445 Cut, paste, cut, paste till it can separate those two double 378 00:19:06,445 --> 00:19:08,520 helices from each other. 379 00:19:08,520 --> 00:19:13,200 Remarkably, this enzyme is called topoisomerase. 380 00:19:18,980 --> 00:19:26,750 This job is done by topoisomerase, actually, by 381 00:19:26,750 --> 00:19:28,750 topoisomerase II. 382 00:19:28,750 --> 00:19:30,820 There's a couple of different topoisomerases, and it's 383 00:19:30,820 --> 00:19:34,740 topoisomerase II that does this particular job, cuts and 384 00:19:34,740 --> 00:19:37,680 seals up that double-stranded break. 385 00:19:37,680 --> 00:19:40,080 All right. 386 00:19:40,080 --> 00:19:42,250 It is amazing how this works. 387 00:19:42,250 --> 00:19:47,080 Let's take another problem in how we do DNA replication. 388 00:19:47,080 --> 00:19:53,900 So let's deal with fidelity. 389 00:19:53,900 --> 00:19:57,305 The fidelity, accuracy of replication. 390 00:20:05,100 --> 00:20:07,810 I have my strand. 391 00:20:07,810 --> 00:20:09,730 Which direction do we go? 392 00:20:09,730 --> 00:20:13,900 We go, for this template, five prime to three prime. 393 00:20:13,900 --> 00:20:16,550 This way goes five prime to three prime, the opposite 394 00:20:16,550 --> 00:20:17,890 direction there. 395 00:20:17,890 --> 00:20:21,040 I now add on. 396 00:20:21,040 --> 00:20:22,790 If this is a T, what do I add in? 397 00:20:22,790 --> 00:20:23,996 AUDIENCE: [INAUDIBLE]. 398 00:20:23,996 --> 00:20:31,000 ERIC LANDER: If it's a GCGTAAT, et cetera. 399 00:20:31,000 --> 00:20:32,740 Why does the right base go in? 400 00:20:37,540 --> 00:20:38,864 Why does the right base go in? 401 00:20:38,864 --> 00:20:39,318 Yeah? 402 00:20:39,318 --> 00:20:40,680 AUDIENCE: Hydrogen bonding. 403 00:20:40,680 --> 00:20:41,030 ERIC LANDER: Hydrogen bonding. 404 00:20:41,030 --> 00:20:42,880 It's got that these hydrogen bonds. 405 00:20:42,880 --> 00:20:44,440 AT makes two hydrogen bonds. 406 00:20:44,440 --> 00:20:46,610 GC makes three hydrogen bonds. 407 00:20:46,610 --> 00:20:50,500 The wrong base could never go in. 408 00:20:50,500 --> 00:20:50,800 Sorry. 409 00:20:50,800 --> 00:20:54,160 In biochemistry, do you ever say never? 410 00:20:54,160 --> 00:20:56,400 No, we say K equilibrium. 411 00:20:56,400 --> 00:20:59,940 We say how much more unfavored is it for the 412 00:20:59,940 --> 00:21:02,350 wrong base to go in? 413 00:21:02,350 --> 00:21:05,500 It's not impossible, it's just disfavored, because it's 414 00:21:05,500 --> 00:21:08,260 energetically less good. 415 00:21:08,260 --> 00:21:10,940 How much energetically less good is it? 416 00:21:10,940 --> 00:21:14,660 What is the delta G for putting in the wrong base? 417 00:21:14,660 --> 00:21:15,910 It's not infinity. 418 00:21:18,470 --> 00:21:24,660 It turns out that there is an equilibrium constant for 419 00:21:24,660 --> 00:21:33,260 putting in the wrong base, and that is K equilibrium is about 420 00:21:33,260 --> 00:21:37,280 10 to the third for the right base, 10 to the minus third 421 00:21:37,280 --> 00:21:39,590 for the wrong base. 422 00:21:39,590 --> 00:21:40,290 Thank goodness. 423 00:21:40,290 --> 00:21:43,930 So only one time in 1,000 does it put in the wrong base. 424 00:21:43,930 --> 00:21:45,130 That's what that has to mean, right? 425 00:21:45,130 --> 00:21:48,870 If it's 1,000 times less favored energetically, it 426 00:21:48,870 --> 00:21:55,410 means you only make a mistake one letter in 1,000. 427 00:21:55,410 --> 00:21:57,700 How do you feel about that for your own genomes? 428 00:21:57,700 --> 00:21:59,440 Is that a level of quality control you 429 00:21:59,440 --> 00:22:00,500 are satisfied with? 430 00:22:00,500 --> 00:22:01,226 AUDIENCE: No. 431 00:22:01,226 --> 00:22:02,180 ERIC LANDER: No. 432 00:22:02,180 --> 00:22:04,980 How big is a typical gene? 433 00:22:04,980 --> 00:22:08,220 Typical gene is, in terms of its protein coding 434 00:22:08,220 --> 00:22:10,708 information, you guys already know about DNA goes to RNA 435 00:22:10,708 --> 00:22:11,090 goes to protein. 436 00:22:11,090 --> 00:22:13,870 It's about 2,000 bases of protein coding information. 437 00:22:13,870 --> 00:22:18,870 That guarantees two mistakes per cell division. 438 00:22:18,870 --> 00:22:20,310 Not good. 439 00:22:20,310 --> 00:22:22,320 Two mistakes per cell division. 440 00:22:22,320 --> 00:22:23,110 That's not OK. 441 00:22:23,110 --> 00:22:26,340 That's two mistakes per cell division. 442 00:22:26,340 --> 00:22:31,030 That would be two errors per cell division, and you have a 443 00:22:31,030 --> 00:22:35,440 lot of cell divisions, you're in a lot of trouble. 444 00:22:35,440 --> 00:22:38,200 So it turns out something more is needed. 445 00:22:38,200 --> 00:22:41,030 Quality control is needed. 446 00:22:41,030 --> 00:22:49,770 So later, it was discovered that the enzyme DNA 447 00:22:49,770 --> 00:23:01,350 polymerase, which has a five prime to three prime 448 00:23:01,350 --> 00:23:08,200 polymerization activity also does a second thing. 449 00:23:12,680 --> 00:23:16,590 That same enzyme, DNA polymerase, is also a three 450 00:23:16,590 --> 00:23:19,590 prime to five prime exonuclease. 451 00:23:22,360 --> 00:23:25,120 What do you think an exonuclease is? 452 00:23:25,120 --> 00:23:26,000 AUDIENCE: [INAUDIBLE]. 453 00:23:26,000 --> 00:23:28,320 ERIC LANDER: Take stuff out. 454 00:23:28,320 --> 00:23:31,740 So it adds bases in the forward direction, but it also 455 00:23:31,740 --> 00:23:34,910 goes backwards and takes bases out. 456 00:23:34,910 --> 00:23:36,710 Isn't that dumb? 457 00:23:36,710 --> 00:23:38,220 I thought we were trying to synthesize, but we're also 458 00:23:38,220 --> 00:23:39,470 unsynthesizing. 459 00:23:41,410 --> 00:23:44,598 With some probability, it goes backwards and takes out bases. 460 00:23:48,590 --> 00:23:53,290 Turns out that the probability of taking out a base backwards 461 00:23:53,290 --> 00:23:54,860 is higher if it's the wrong base. 462 00:23:58,840 --> 00:24:02,540 It's proofreading as it goes as I hope you are. 463 00:24:02,540 --> 00:24:05,440 It's proofreading. 464 00:24:05,440 --> 00:24:09,160 It goes backwards and takes bases out more often. 465 00:24:09,160 --> 00:24:12,400 Sometimes it takes out the right bases, but it is 466 00:24:12,400 --> 00:24:14,170 proofreading its work. 467 00:24:20,230 --> 00:24:23,080 And more often when it's the wrong base, it goes backwards, 468 00:24:23,080 --> 00:24:25,920 and so you get the benefit of a K equilibrium from the 469 00:24:25,920 --> 00:24:26,940 original base. 470 00:24:26,940 --> 00:24:29,420 And then there's a separate K equilibrium for the 471 00:24:29,420 --> 00:24:30,985 proofreading, and that helps you. 472 00:24:30,985 --> 00:24:35,260 And when you combine the proofreading with the original 473 00:24:35,260 --> 00:24:42,520 accuracy, now, we're down to something like 10 to the minus 474 00:24:42,520 --> 00:24:48,070 five or 10 to the minus six errors per 475 00:24:48,070 --> 00:24:49,760 base, per cell division. 476 00:24:53,510 --> 00:24:55,935 It's only making on the order of one error per million. 477 00:25:00,000 --> 00:25:03,080 Now are we satisfied? 478 00:25:03,080 --> 00:25:03,730 No. 479 00:25:03,730 --> 00:25:05,370 You guys pretty hard nosed. 480 00:25:05,370 --> 00:25:09,760 Not good enough, because you have 50 cell divisions to make 481 00:25:09,760 --> 00:25:11,340 more and some cells go through many, many, 482 00:25:11,340 --> 00:25:12,880 many more cell divisions. 483 00:25:12,880 --> 00:25:14,400 Not acceptable. 484 00:25:14,400 --> 00:25:15,650 But it's a start. 485 00:25:20,430 --> 00:25:22,940 So proofreading helps. 486 00:25:22,940 --> 00:25:26,850 So we have the fidelity of replication. 487 00:25:26,850 --> 00:25:31,120 Replication makes an error at a rate of 10 488 00:25:31,120 --> 00:25:32,650 to the minus third. 489 00:25:32,650 --> 00:25:39,100 Proofreading brings you down to 10 to the minus six, and 490 00:25:39,100 --> 00:25:41,130 there's another process. 491 00:25:41,130 --> 00:25:44,400 There are a set of enzymes that go around and feel the 492 00:25:44,400 --> 00:25:48,340 DNA double helix after it's finished, and if you put in 493 00:25:48,340 --> 00:25:53,620 the wrong base, the width of the helix is not right. 494 00:25:53,620 --> 00:25:55,550 The shape is wrong. 495 00:25:55,550 --> 00:25:57,900 It feels for mismatches. 496 00:25:57,900 --> 00:26:00,060 So there is a mismatch repair system. 497 00:26:04,980 --> 00:26:10,900 Mismatch repair comes along, and if there was an error 498 00:26:10,900 --> 00:26:15,120 right here, the helix bulges out too much let's say. 499 00:26:15,120 --> 00:26:22,400 Mismatch repair cuts, removes some DNA, and gives the cell 500 00:26:22,400 --> 00:26:25,510 another chance to do it again. 501 00:26:25,510 --> 00:26:30,350 Mismatch repair gets you down to something in the 502 00:26:30,350 --> 00:26:33,020 neighborhood of 10 to the minus eighth, 10 503 00:26:33,020 --> 00:26:35,840 to the minus ninth. 504 00:26:35,840 --> 00:26:37,200 Let's say for the sake of argument, 10 505 00:26:37,200 --> 00:26:38,390 to the minus ninth. 506 00:26:38,390 --> 00:26:41,610 You're genome is about three times 10 to the ninth. 507 00:26:41,610 --> 00:26:45,100 Now making that's one or two errors per genome, 508 00:26:45,100 --> 00:26:46,350 that's not so bad. 509 00:26:49,540 --> 00:26:51,240 Why do we care? 510 00:26:51,240 --> 00:26:52,890 Why am I bothering you with this? 511 00:26:52,890 --> 00:26:55,810 Who cares between 10 to minus sixth, 10 to the minus ninth? 512 00:26:55,810 --> 00:26:57,060 Big deal. 513 00:26:59,630 --> 00:27:07,450 Well, a few percent of you in this class are heterozygous 514 00:27:07,450 --> 00:27:11,590 for a mutation in the mismatch repair enzymes. 515 00:27:11,590 --> 00:27:12,730 Don't worry. 516 00:27:12,730 --> 00:27:16,000 Your cells have the other copy that's good. 517 00:27:16,000 --> 00:27:20,930 But suppose one of your cells were to lose, by mutation, the 518 00:27:20,930 --> 00:27:23,060 good copy of the mismatch repair enzyme? 519 00:27:23,060 --> 00:27:26,250 And now that cell in your body had no copies of mismatch 520 00:27:26,250 --> 00:27:27,500 repair enzyme. 521 00:27:31,780 --> 00:27:33,250 What do you think is going to happen to your DNA 522 00:27:33,250 --> 00:27:35,930 replication? 523 00:27:35,930 --> 00:27:37,660 Instead of being one in a billion, it would be one in a 524 00:27:37,660 --> 00:27:40,220 million accuracy. 525 00:27:40,220 --> 00:27:41,460 Turns out you have an extremely high 526 00:27:41,460 --> 00:27:44,780 risk of colon cancer. 527 00:27:44,780 --> 00:27:47,660 There are hereditary colon cancer syndromes that are due 528 00:27:47,660 --> 00:27:50,800 to inherited defects in the mismatch repair system. 529 00:27:50,800 --> 00:27:53,380 It is not at all trivial. 530 00:27:53,380 --> 00:27:55,900 Hereditary polyposis coli is due to a 531 00:27:55,900 --> 00:27:57,340 defect in this enzyme. 532 00:28:02,770 --> 00:28:03,720 It matters. 533 00:28:03,720 --> 00:28:06,010 You've got to get it down to that level because otherwise, 534 00:28:06,010 --> 00:28:09,780 you're getting mutations that cause cancer, that is, when 535 00:28:09,780 --> 00:28:12,430 you lose both copies, if you lost both copies. 536 00:28:12,430 --> 00:28:14,890 Most of your cells would be fine, but if you'd lose the 537 00:28:14,890 --> 00:28:18,100 other good copy, by chance, that cell can 538 00:28:18,100 --> 00:28:21,490 go on to cause cancer. 539 00:28:21,490 --> 00:28:23,460 So this stuff actually matters. 540 00:28:23,460 --> 00:28:29,120 Finally, finally, speed. 541 00:28:29,120 --> 00:28:31,900 Kind of fun to talk about speed. 542 00:28:31,900 --> 00:28:35,291 How fast does polymerase work? 543 00:28:35,291 --> 00:28:43,720 It turns out that polymerase is able to polymerize 2,000 544 00:28:43,720 --> 00:28:50,180 nucleotides per second. 545 00:28:50,180 --> 00:28:52,070 That's very impressive to me. 546 00:28:52,070 --> 00:28:54,910 It zips along at 2,000 nucleotides per second, 547 00:28:54,910 --> 00:28:59,050 installing the right base, getting it right only 99.9% of 548 00:28:59,050 --> 00:29:03,260 the time, proofreading as it goes, and gets the whole thing 549 00:29:03,260 --> 00:29:06,580 done 2,000 letters in a second. 550 00:29:06,580 --> 00:29:08,580 That is impressive engineering. 551 00:29:08,580 --> 00:29:12,110 That is really impressive engineering. 552 00:29:12,110 --> 00:29:17,170 So that's kind of how DNA replication works well, except 553 00:29:17,170 --> 00:29:18,420 for one thing. 554 00:29:29,540 --> 00:29:30,790 Kornberg was a biochemist. 555 00:29:33,290 --> 00:29:35,300 Biochemists purify things in test tubes. 556 00:29:41,180 --> 00:29:49,650 He discovered an enzyme, Kornberg's polymerase. 557 00:29:54,880 --> 00:29:59,420 How do we know it's the enzyme the cell actually 558 00:29:59,420 --> 00:30:03,070 uses to copy its DNA? 559 00:30:03,070 --> 00:30:04,600 See, I'm a geneticist. 560 00:30:04,600 --> 00:30:07,300 I look at Kornberg and I say, nice job. 561 00:30:07,300 --> 00:30:12,030 You showed me an enzyme that in a test tube is capable of 562 00:30:12,030 --> 00:30:14,250 polymerizing DNA. 563 00:30:14,250 --> 00:30:17,340 How do I know that's the enzyme that's actually doing 564 00:30:17,340 --> 00:30:19,110 it from the cell copies its whole genome? 565 00:30:22,210 --> 00:30:25,858 What does a geneticist want to see? 566 00:30:25,858 --> 00:30:27,350 AUDIENCE: A mutant. 567 00:30:27,350 --> 00:30:28,230 ERIC LANDER: A mutant. 568 00:30:28,230 --> 00:30:32,000 Show me a mutant then I'll believe. 569 00:30:32,000 --> 00:30:39,330 So someone went along and took E. colis one at a time because 570 00:30:39,330 --> 00:30:40,400 what else could they do. 571 00:30:40,400 --> 00:30:43,670 And for every single E. coli they grew up from a plate, 572 00:30:43,670 --> 00:30:45,722 they purified Kornberg's enzyme. 573 00:30:45,722 --> 00:30:48,680 And you know what they found? 574 00:30:48,680 --> 00:30:55,710 They found a mutant E. coli that lacked Kornberg's enzyme, 575 00:30:55,710 --> 00:30:57,900 and it could replicate its DNA just fine. 576 00:31:01,950 --> 00:31:03,200 What does that tell us? 577 00:31:07,440 --> 00:31:11,370 Kornberg actually had the wrong enzyme. 578 00:31:11,370 --> 00:31:13,270 He still deserves a Nobel Prize for it because he got an 579 00:31:13,270 --> 00:31:15,340 enzyme that could copy DNA. 580 00:31:15,340 --> 00:31:18,790 It's actually not the main enzyme that does the job. 581 00:31:18,790 --> 00:31:22,270 Because we can make a mutant that lacks that enzyme and it 582 00:31:22,270 --> 00:31:27,220 can still copy the DNA, it can't be the main enzyme. 583 00:31:27,220 --> 00:31:31,320 Turns out what Kornberg found was a minor polymerase that 584 00:31:31,320 --> 00:31:34,320 was used in those mismatch repair situations that would 585 00:31:34,320 --> 00:31:37,190 come along and do the tidying and clean up. 586 00:31:37,190 --> 00:31:39,810 The main enzyme turned out to be another enzyme, a more 587 00:31:39,810 --> 00:31:41,390 complicated enzyme. 588 00:31:41,390 --> 00:31:45,450 So my point about biochemistry and genetics both having to 589 00:31:45,450 --> 00:31:48,900 talk to each other, you only really know something when you 590 00:31:48,900 --> 00:31:51,940 have it from a biochemical point of view and the genetic 591 00:31:51,940 --> 00:31:53,000 point of view. 592 00:31:53,000 --> 00:31:54,500 The two have to go together. 593 00:31:54,500 --> 00:31:56,110 Kornberg's enzyme is a great enzyme, 594 00:31:56,110 --> 00:31:57,470 it's a fantastic enzyme. 595 00:31:57,470 --> 00:32:00,410 It just happens not to be the main enzyme, and you can only 596 00:32:00,410 --> 00:32:02,560 know that by genetics. 597 00:32:02,560 --> 00:32:05,360 Of course, you can only purify it by biochemistry. 598 00:32:05,360 --> 00:32:06,050 All right. 599 00:32:06,050 --> 00:32:09,910 So that's DNA replication. 600 00:32:09,910 --> 00:32:12,640 Any questions about DNA replication before I go on? 601 00:32:12,640 --> 00:32:13,060 Yes? 602 00:32:13,060 --> 00:32:15,210 AUDIENCE: [INAUDIBLE]. 603 00:32:15,210 --> 00:32:17,450 ERIC LANDER: Polymerase III or polymerase II, depending on 604 00:32:17,450 --> 00:32:18,200 the organism. 605 00:32:18,200 --> 00:32:19,330 They're all called polymerases. 606 00:32:19,330 --> 00:32:20,650 They're all DNA polymerases. 607 00:32:20,650 --> 00:32:22,470 They just get different names and numbers. 608 00:32:22,470 --> 00:32:25,150 Turns out most cells have multiple polymerases and 609 00:32:25,150 --> 00:32:27,780 Kornberg found the kind of simpler polymerase. 610 00:32:27,780 --> 00:32:29,980 The main replication polymerase also called 611 00:32:29,980 --> 00:32:32,190 polymerase but with a different number, is a 612 00:32:32,190 --> 00:32:33,410 different more complicated enzyme. 613 00:32:33,410 --> 00:32:33,800 Yes? 614 00:32:33,800 --> 00:32:39,620 AUDIENCE: How does the enzyme know which one is the right..? 615 00:32:39,620 --> 00:32:41,520 ERIC LANDER: how does it know which one is right? 616 00:32:41,520 --> 00:32:44,747 AUDIENCE: [INAUDIBLE]. 617 00:32:44,747 --> 00:32:47,080 ERIC LANDER: Because 50% of the time you get it wrong. 618 00:32:47,080 --> 00:32:48,530 Do you know what bacteria do? 619 00:32:48,530 --> 00:32:49,420 What a great question. 620 00:32:49,420 --> 00:32:50,670 How would it know which one to get right? 621 00:32:53,500 --> 00:32:56,400 Know what bacteria do? 622 00:32:56,400 --> 00:32:57,780 They're very tricky. 623 00:32:57,780 --> 00:33:00,210 They mark their DNA, don't worry about this. 624 00:33:00,210 --> 00:33:02,680 They mark their DNA with methyl groups. 625 00:33:02,680 --> 00:33:04,870 There is an enzyme that comes along and put methyl groups at 626 00:33:04,870 --> 00:33:11,640 certain positions, but that enzyme is kind of slow. 627 00:33:11,640 --> 00:33:15,100 So I have a methyl-marked DNA double helix. 628 00:33:15,100 --> 00:33:18,580 When I replicate it, the new strand is made, and what does 629 00:33:18,580 --> 00:33:19,610 the new strand lack? 630 00:33:19,610 --> 00:33:21,000 AUDIENCE: Little methyl groups. 631 00:33:21,000 --> 00:33:22,910 ERIC LANDER: Little methyl groups. 632 00:33:22,910 --> 00:33:25,110 It'll get them eventually because that slow enzyme will 633 00:33:25,110 --> 00:33:29,050 come along and put them on, but mismatch repair is fast. 634 00:33:29,050 --> 00:33:31,940 So what is mismatch repair looking for? 635 00:33:31,940 --> 00:33:34,480 The little methyl groups that are kind of breadcrumbs that 636 00:33:34,480 --> 00:33:38,330 say, this was the old strand, and this guy is the new stand. 637 00:33:38,330 --> 00:33:39,370 It's thought of everything. 638 00:33:39,370 --> 00:33:41,020 It's really smart. 639 00:33:41,020 --> 00:33:42,270 Very, very smart.