0:00:00.969,0:00:10.100 The topic of today's lecture is DNA function: replication as a molecular basis of heredity. 0:00:10.100,0:00:17.570 The lecture is part of Module 1: Animal Genetics, that is a part of the ISAGREED project. 0:00:17.570,0:00:26.140 This presentation was supported by Erasmus+ KA2 Cooperation Partnerships Grant "Innovation 0:00:26.140,0:00:33.739 of the content and structure of study programmes in the field of management of animal genetic 0:00:33.739,0:00:36.579 and food resources using digitalization". 0:00:36.579,0:00:46.140 As part of the lecture, we will first explain the concept of replication and its importance, 0:00:46.140,0:00:54.970 we will talk about proposed replication models and the enzymes catalyzing this reaction. 0:00:54.970,0:01:03.410 We will then explain in detail the so-called semi-conservative mechanism of replication. 0:01:03.410,0:01:13.280 DNA replication is the process of making copies of a deoxyribonucleic acid molecule, thereby 0:01:13.280,0:01:20.030 transferring genetic information from one DNA molecule to another molecule of the same 0:01:20.030,0:01:21.909 type. 0:01:21.909,0:01:28.869 It has to occur before cell division (mitosis or meiosis) and is the basic assumption that 0:01:28.869,0:01:35.159 each daughter cell receives DNA typical for the given species. 0:01:35.159,0:01:43.130 During division, a cell must copy its entire genome so that both daughter cells carry the 0:01:43.130,0:01:45.630 same information. 0:01:45.630,0:01:53.579 You will learn about subsequent events, which are transcription and translation, in other 0:01:53.579,0:01:55.909 lectures. 0:01:55.909,0:02:02.170 You have already heard about the structure of DNA in the previous lecture, so I will 0:02:02.170,0:02:10.099 only briefly mention that DNA is composed of two complementary polynucleotide strands 0:02:10.099,0:02:14.310 twisted into a so-called double helix. 0:02:14.310,0:02:21.160 These strands are connected by hydrogen bonds based on the complementarity between the nitrogen 0:02:21.160,0:02:28.360 bases, where adenine is always connected to thymine and cytosine to guanine. 0:02:28.360,0:02:35.800 The complementarity ensures that a newly synthesized strand will correspond to the original one. 0:02:35.800,0:02:43.470 We distinguish the so-called 3 prime and 5 prime end on each strand. 0:02:43.470,0:02:50.170 The strands are oriented antiparallel to each other, which means that opposite the 5 prime 0:02:50.170,0:03:00.440 end of one strand lies the 3 prime end of the other strand and vice versa. 0:03:00.440,0:03:10.300 DNA replication happens in a so-called semi-conservative mechanism, meaning that each newly created 0:03:10.300,0:03:20.430 molecule consists of one strand of the original DNA molecule, the so-called template, and 0:03:20.430,0:03:27.340 one strand newly synthesized according to base complementarity. 0:03:27.340,0:03:35.700 Both strands of the original DNA molecule can serve as a template or matrix. 0:03:35.700,0:03:43.470 In the past, the so-called conservative replication model was also proposed, in which both daughter 0:03:43.470,0:03:52.019 DNA strands are newly synthesized, and both template strands of the parent molecule are 0:03:52.019,0:03:54.170 left in their original composition. 0:03:54.170,0:04:02.750 However, this model is currently being overcome, similar to the model of so-called dispersive 0:04:02.750,0:04:11.970 replication, which produces two helices in which each strand contains alternating segments 0:04:11.970,0:04:15.110 of old and new DNA. 0:04:15.110,0:04:19.799 Several enzymes catalyze replication. 0:04:19.799,0:04:29.380 They are primarily DNA polymerases that catalyze the synthesis of the complementary DNA strand 0:04:29.380,0:04:36.430 from deoxyribonucleotides on the DNA matrix strand. 0:04:36.430,0:04:41.080 This ability is called endonuclease activity. 0:04:41.080,0:04:47.910 Polymerization occurs from the 5 prime end to the 3 prime end. 0:04:47.910,0:04:57.520 For their activity, DNA polymerases need a short oligonucleotide, the so-called primer, 0:04:57.520,0:05:04.070 from the 3 prime end of which the synthesis is started. 0:05:04.070,0:05:13.449 Some polymerases also have exonuclease activity, which means removing nucleotides from the 0:05:13.449,0:05:17.600 end of the daughter strand. 0:05:17.600,0:05:26.169 Exonuclease activity is necessary for removing RNA primers used in DNA replication and is 0:05:26.169,0:05:31.080 called proof-reading or control activity. 0:05:31.080,0:05:38.819 It is also important if the wrong nucleotide is included as it allows a step back and correction 0:05:38.819,0:05:42.990 - including the correct (complementary) base. 0:05:42.990,0:05:50.270 Therefore, proof-reading activity reduces the frequency of spontaneous mutations caused 0:05:50.270,0:06:00.870 by the DNA polymerase wrong activity, estimated at an average of one error per 10 to power 0:06:00.870,0:06:04.130 of 7 replicated base pairs. 0:06:04.130,0:06:10.270 Three types of DNA polymerases are known in prokaryotic cells. 0:06:10.270,0:06:18.090 DNA polymerase I has the function of polymerization as well as exonuclease activity. 0:06:18.090,0:06:26.919 DNA polymerase II has exonuclease activity and plays a role in polymerization termination. 0:06:26.919,0:06:34.360 DNA polymerase III is a so-called holoenzyme. 0:06:34.360,0:06:42.060 It has three subunits with multiple functions, which combine for better efficiency (or processivity) 0:06:42.060,0:06:46.680 into a dimer of twice three subunits. 0:06:46.680,0:06:58.879 Together with other proteins, it recognizes the RNA primer complex with the DNA template 0:06:58.879,0:07:01.449 strand. 0:07:01.449,0:07:09.300 It polymerizes at a speed of approximately thirty thousand nucleotides per minute. 0:07:09.300,0:07:18.509 Other enzymes in the replication process include DNA helicases, which catalyze the unwinding 0:07:18.509,0:07:24.169 of the DNA strands of the helix by disrupting hydrogen bonds. 0:07:24.169,0:07:33.240 Another enzyme is primase, which catalyzes the synthesis of an RNA primer (oligoribonucleotide) 0:07:33.240,0:07:43.129 from the 3 prime end of which a short polydeoxyribunucleotide is synthesized. 0:07:43.129,0:07:49.510 This complex is called the Okazaki fragment - it will be explained later. 0:07:49.510,0:07:59.659 The following enzyme is DNA ligase, which catalyzes the joining of polynucleotides. 0:07:59.659,0:08:05.850 It plays a role in connecting Okazaki fragments into a continuous strand. 0:08:05.850,0:08:14.550 As already mentioned, DNA replication takes place in a so-called semi-conservative mechanism, 0:08:14.550,0:08:23.379 which means that both strands of the original DNA can serve as templates for synthesizing 0:08:23.379,0:08:25.090 new (daughter) strands. 0:08:25.090,0:08:34.500 New DNA is, therefore, always made up of one original strand and one newly synthesized 0:08:34.500,0:08:40.409 strand based on the complementarity of the nucleotide bases. 0:08:40.409,0:08:49.510 The DNA polymerase moves along the template chain from the 3 prime to the 5 prime end, 0:08:49.510,0:08:59.410 and new strands are created in the opposite direction, that means from the 5 prime to 0:08:59.410,0:09:02.660 the 3 prime end. 0:09:02.660,0:09:08.380 Here I would like to remind you again of the importance of complementary base pairing, 0:09:08.380,0:09:13.740 which is necessary for the newly entering daughter's DNA to match the original parental 0:09:13.740,0:09:14.740 DNA. 0:09:14.740,0:09:24.290 The picture shows that cytosine always bonds with guanine with three hydrogen bonds, while 0:09:24.290,0:09:28.589 adenine with thymine with two hydrogen bonds. 0:09:28.589,0:09:39.160 This complementarity is an essential prerequisite for preserving and transmitting genetic information. 0:09:39.160,0:09:46.510 Self-replication begins with the denaturation of double-helix DNA when initiation proteins 0:09:46.510,0:09:53.519 that bind to the DNA unfold its structure by breaking hydrogen bonds. 0:09:53.519,0:10:00.269 The places where the DNA structure is first disrupted are called origins of replication 0:10:00.269,0:10:06.910 and are determined by a unique nucleotide sequence. 0:10:06.910,0:10:13.600 The enzyme DNA helicase catalyzes the process of breaking the double helix structure of 0:10:13.600,0:10:14.950 DNA. 0:10:14.950,0:10:23.210 Y-shaped formations called replication forks are typical for replication origins. 0:10:23.210,0:10:31.290 At one origin of replication, two forks are formed that move away from each other, which 0:10:31.290,0:10:35.779 is why replication is referred to as bidirectional. 0:10:35.779,0:10:45.350 Once the initiation proteins are bound to the DNA and open its double helix structure, 0:10:45.350,0:10:50.730 a group of proteins binds to the origin of replication. 0:10:50.730,0:10:56.579 It cooperates in synthesizing a new strand of DNA. 0:10:56.579,0:11:04.020 These are, for example, so-called single-strand binding proteins, which protect single-stranded 0:11:04.020,0:11:10.690 DNA released by helicase from re-pairing. 0:11:10.690,0:11:18.520 Since DNA polymerase (the main enzyme catalyzing replication) cannot start synthesizing a new 0:11:18.520,0:11:26.709 strand, there must be another enzyme that can join two free nucleotides and start synthesizing 0:11:26.709,0:11:30.660 a new strand according to single-stranded DNA. 0:11:30.660,0:11:38.860 This enzyme is called primase and forms short stretches of approximately ten nucleotides 0:11:38.860,0:11:43.200 in length, referred to as primers. 0:11:43.200,0:11:51.040 However, these are not sections of DNA, but sections of a similar molecule known as ribonucleic 0:11:51.040,0:11:56.380 acid, hence the so-called RNA primers. 0:11:56.380,0:12:05.850 DNA polymerase can already extend these RNA primers as a new DNA strand. 0:12:05.850,0:12:16.970 Later, the RNA primers are removed thanks to the exonuclease activity of the DNA polymerase. 0:12:16.970,0:12:27.380 Since DNA can only be synthesized in the 5 prime – 3 prime end direction, a particular 0:12:27.380,0:12:31.500 problem occurs at the replication fork. 0:12:31.500,0:12:38.330 The replication fork is asymmetric since the strands are in opposite orientations in the 0:12:38.330,0:12:40.470 original double helix. 0:12:40.470,0:12:50.480 One new strand is synthesized at the replication fork according to the template in the 3 prime 0:12:50.480,0:12:52.880 → 5 prime end direction. 0:12:52.880,0:12:58.649 (A 5 prime → 3 prime end strand is formed). 0:12:58.649,0:13:05.320 This strand that is formed continuously is called a leading strand. 0:13:05.320,0:13:11.699 The second new strand is synthesized at the replication fork according to the template 0:13:11.699,0:13:16.240 in the 5 prime → 3 prime end direction. 0:13:16.240,0:13:23.300 However, no DNA polymerase can extend the 5 prime end of DNA. 0:13:23.300,0:13:33.670 Therefore, it grows discontinuously in this direction, meaning short sections of DNA (so-called 0:13:33.670,0:13:42.610 Okazaki fragments) are synthesized in the direction 5 prime → 3 prime, which are joined 0:13:42.610,0:13:47.050 into a continuous strand later. 0:13:47.050,0:13:55.270 The so-called lagging strand is then made up of many separate sections of DNA, the Okazaki 0:13:55.270,0:13:56.480 fragments. 0:13:56.480,0:14:03.770 The following enzymes are needed to create a continuous DNA strand from Okazaki fragments: 0:14:03.770,0:14:16.009 DNA polymerase I, which removes RNA primers and replaces RNA primers with DNA, and DNA 0:14:16.009,0:14:22.610 ligase, which finally joins all the sections together. 0:14:22.610,0:14:29.519 This strand that is formed discontinuously is called a lagging strand. 0:14:29.519,0:14:35.750 Replication of the prokaryotic chromosome ends at specific sequences, the so-called 0:14:35.750,0:14:44.410 replication terminators (TER), to which a protein inhibiting helicase activity binds 0:14:44.410,0:14:47.480 and thereby stops the formation of the replication fork. 0:14:47.480,0:14:56.910 DNA replication in eukaryotic organisms is similar to prokaryotes. 0:14:56.910,0:14:59.810 However, it is more complicated. 0:14:59.810,0:15:08.430 For example, replication at the ends of linear molecules - telomeres constitutes a specific 0:15:08.430,0:15:17.389 problem that is solved by the RNA-containing enzyme telomerase. 0:15:17.389,0:15:27.889 This picture again shows a model of the entire replisome – the unwound DNA and protein 0:15:27.889,0:15:32.870 machinery at the replication fork. 0:15:32.870,0:15:39.540 In conclusion, we will summarize the most important findings from this lecture. 0:15:39.540,0:15:48.360 DNA replication occurs by a semi-conservative mechanism, where each original DNA strand 0:15:48.360,0:15:53.190 is a template for creating a new molecule. 0:15:53.190,0:15:59.069 It is a process catalyzed by several enzymes. 0:15:59.069,0:16:06.459 The important thing to remember is DNA polymerases, in particular. 0:16:06.459,0:16:15.251 The polymerization of the new strand always takes place only in the 5 prime – 3 prime 0:16:15.251,0:16:17.180 direction. 0:16:17.180,0:16:29.990 DNA polymerase needs short stretches, RNA primers, to initiate synthesis. 0:16:29.990,0:16:37.160 Synthesis of the new strand is continuous on one template strand (this is the so-called 0:16:37.160,0:16:44.440 leading strand) and discontinuous on the other with the formation of so-called Okazaki fragments 0:16:44.440,0:16:49.160 (the so-called lagging strand). 0:16:49.160,0:16:58.800 In eukaryotes, replication occurs in the S-phase of cell division, starting at many locations. 0:16:58.800,0:17:06.660 Replication is essential for preserving genetic information and is the essence of heredity 0:17:06.660,0:17:10.890 at the molecular level. 0:17:10.890,0:17:14.839 At this moment I would like to thank you for your attention. 0:17:14.839,0:17:19.289 If you have any questions, you can use the email listed here.