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Www.soran.edu.iq Cell and Molecular Biology Behrouz Mahmoudi DNA Replication 1.

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1 www.soran.edu.iq Cell and Molecular Biology Behrouz Mahmoudi DNA Replication 1

2 www.soran.edu.iq Semiconservative DNA Replication 1. Watson and Crick DNA model implies a mechanism for replication: a. Unwind the DNA molecule. b. Separate the two strands. c. Make a complementary copy for each strand. 2

3 www.soran.edu.iq 2.Three possible models were proposed for DNA replication: a. Conservative model proposed both strands of one copy would be entirely old DNA, while the other copy would have both strands of new DNA. b. Dispersive model was that dsDNA might fragment, replicate dsDNA, and then reassemble, creating a mosaic of old and new dsDNA regions in each new chromosome. c. Semiconservative model is that DNA strands separate, and a complementary strand is synthesized for each, so that sibling chromatids have one old and one new strand. This model was the winner in the Meselson and Stahl experiment. 3

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5 The Meselson-Stahl Experiment 1.Meselson and Stahl (1958) grew E. coli in a heavy (not radioactive) isotope of nitrogen, 15 N in the form of 15 NH 4 Cl. Because it is heavier, DNA containing 15 N is more dense than DNA with normal 14 N, and so can be separated by CsCl density gradient centrifugation (Box 3.1). 2.Once the E. coli were labeled with heavy 15 N, the researchers shifted the cells to medium containing normal 14 N, and took samples at time points. DNA was extracted from each sample and analyzed in CsCl density gradients (Figure 3.2). 5

6 www.soran.edu.iq Fig. 3.2 The Meselson-Stahl experiment, which showed that DNA replicates semiconservatively 6

7 www.soran.edu.iq Box Fig. 3.1 Equilibrium centrifugation of DNA of different densities in a cesium chloride density gradient 7

8 www.soran.edu.iq 3. After one replication cycle in normal 14 N medium, all DNA had density intermediate between heavy and normal. After two replication cycles, there were two bands in the density gradient, one at the intermediate position, and one at the position for DNA containing entirely 14 N. 4. Results compared with the three proposed models: a. Does not fit conservative model, because after one generation there is a single intermediate band, rather than one with entirely 15 N DNA and another with entirely 14 N DNA. b. The dispersive model predicted that a single band of DNA of intermediate density would be present in each generation, gradually becoming less dense as increasing amounts of 14 N were incorporated with each round of replication. Instead, Meselson and Stahl observed two bands of DNA, with the intermediate form decreasing over time. c. The semiconservative model fits the data very well. 8

9 www.soran.edu.iq Roles of DNA Polymerases 1. All DNA polymerases link dNTPs into DNA chains. Main features of the reaction: a. An incoming nucleotide is attached by its 5’-phosphate group to the 3’- OH of the growing DNA chain. Energy comes from the dNTP releasing two phosphates. The DNA chain acts as a primer for the reaction. b. The incoming nucleotide is selected by its ability to hydrogen bond with the complementary base in the template strand. The process is fast and accurate. c. DNA polymerases synthesize only from 5’ to 3’. 2.The enzyme Kornberg isolated was believed to be the only DNA polymerase in E. coli. However, mutations in this gene (polA1) were not lethal, indicating that other DNA polymerases must exist in E. coli. 9

10 www.soran.edu.iq Fig. 3.4a DNA chain elongation catalyzed by DNA polymerase 10

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12 www.soran.edu.iq The properties of DNA polymerases 5.The properties of known E. coli DNA polymerases are: a.DNA polymerase I is a single peptide encoded by polA and used for DNA replication. Replicates DNA in the 5’  3’ direction. Has 5’  3’ exonuclease activity to remove nucleotides from 5’ end of DNA or from an RNA primer. b.DNA polymerase II is a single peptide encoded by polB. Used for DNA repair. c.DNA polymerase III has three polypeptide subunits in the catalytic core of the enzyme: α (encoded by the dnaE gene), ε (dnaQ), and θ (holE). Holoenzyme has an additional six different polypeptides. Replicates DNA in the 5’  3’ direction. d.DNA polymerase IV is encoded by the dinB gene, and is used in DNA repair. e.DNA polymerase V is encoded by umuDC, and is used in DNA repair. 6.E. coli DNA polymerases used for DNA replication (DNA polymerase I and DNA polymerase III) have 3’  5’ exonuclease (proofreading) activity. 12

13 www.soran.edu.iq Molecular Model of DNA Replication 13

14 www.soran.edu.iq Initiation of Replication 1. Replication starts when DNA at the origin of replication denatures to expose the bases, creating a replication fork. Replication is usually bidirectional from the origin. E. coli has one origin, oriC, which has: a.A minimal sequence of about 245 bp required for initiation. b.Three copies of a 13-bp AT-rich sequence. c.Four copies of a 9-bp sequence. 2.Events in E. coli initiating DNA synthesis, derived from in vitro studies: a.Initiator proteins attach. E. coli’s initiator protein is DnaA (from the dnaA gene). b.DNA helicase (from dnaB) binds initiator proteins on the DNA, and denatures the AT-rich region using ATP as an energy source. c.DNA primase (from dnaG) binds helicase to form a primosome, which synthesizes a short (5–10nt) RNA primer.. 14

15 www.soran.edu.iq Fig. 3.5 Model for the formation of a replication bubble at a replication origin in E. coli and the initiation of the new DNA strand 15

16 www.soran.edu.iq Fig. 3.6a, b Model for the events occurring around a single replication fork of the E. coli chromosome 16

17 www.soran.edu.iq Fig. 3.6c-e Model for the events occurring around a single replication fork of the E. coli chromosome 17

18 www.soran.edu.iq 1. When DNA denatures at the oriC, replication forks are formed. DNA replication is usually bi-directional, but will consider events at just one replication fork: a. Single-strand DNA-binding proteins (SSBs) bind the ssDNA formed by helicase, preventing reannealing. b. Primase synthesizes a primer on each template strand. c. DNA polymerase III adds nucleotides to the 3’ end of the primer, synthesizing a new strand complementary to the template, and displacing the SSBs. DNA is made in opposite directions on the two template strands. d. New strand made 5’ → 3’ in same direction as movement of the replication fork is leading strand, while new strand made in opposite direction is lagging strand. Leading strand needs only one primer, while lagging needs a series of primers. 18

19 www.soran.edu.iq 2. Helicase denaturing DNA causes tighter winding in other parts of the circular chromosome. Gyrase relieves this tension. 3. Leading strand is synthesized continuously, while lagging strand is synthesized discontinuously, in the form of Okazaki fragments. DNA replication is therefore semidiscontinuous. 4. Each fragment requires a primer to begin, and is extended by DNA polymerase III. 5. Okazaki data show that these fragments are gradually joined together to make a full-length dsDNA chromosome. DNA polymerase I uses the 3’-OH of the adjacent DNA fragment as a primer, and simultaneously removes the RNA primer while resynthesizing the primer region in the form of DNA. The nick remaining between the two fragments is sealed with DNA ligase. 19

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21 www.soran.edu.iq 6. Key proteins are associated to form a replisome. Template DNA probably bends to allow synthesis of both leading and lagging strands at the replication fork 21

22 www.soran.edu.iq Bidirectional replication of circular DNA molecules 22

23 www.soran.edu.iq Replication of circular DNA and the supercoiling problem 1. Some circular chromosomes (e.g., E. coli) are circular throughout replication, creating a theta-like (θ) shape. As the strands separate on one side of the circle, positive supercoils form elsewhere in the molecule. Replication fork moves about 500 nt/ second, so at 10 bp/turn, replication fork rotates at 3,000 rpm. 2. Topoisomerases relieve the supercoils, allowing the DNA strands to continue separating as the replication forks advance. 23

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25 www.soran.edu.iq 1. Another model for replication is rolling circle(Figure 3.10), which is used by several bacteriophages, including ΦX174 (after a complement is made for the genomic ssDNA) and λ (after circularization by base pairing between the “sticky” ssDNA cos ends) 2. Rolling circle replication begins with a nick (single- stranded break) at the origin of replication. The 5’ end is displaced from the strand, and the 3’ end acts as a primer for DNA polymerase III, which synthesizes a continuous strand using the intact DNA molecule as a template. 3. The 5’ end continues to be displaced as the circle “rolls”, and is protected by SSBs until discontinuous DNA synthesis makes it a dsDNA again. Rolling Circle Replication 25

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27 www.soran.edu.iq 4. A DNA molecule many genomes in length can be made by rolling circle replication. During viral assembly it is cut into individual viral chromosomes and packaged into phage head. 5. Bacteriophage λ, regardless of whether entering the lytic or lysogenic pathway, circularizes its chromosome immediately after infection. a. In a lysogenic infection, the circular DNA integrated into a specific site in the E. coli chromosome by a crossover event. b. In a lytic infection, rolling circle replication produces a long concatamer of λ DNA, and the a viral endonuclease (product of the ter gene) recognize the cos sites and makes the staggered cuts that used to assemble new virus particles. 27

28 www.soran.edu.iq chromosome structure varies at stages of lytic infection of E. coli 28

29 www.soran.edu.iq DNA Replication in Eukaryotes 1.DNA replication is very similar in both prokaryotes and eukaryotes, except that eukaryotes have more than one chromosome. 29

30 www.soran.edu.iq Replicons 1.Eukaryotic chromosomes generally contain much more DNA than those of prokaryotes, and their replication forks move much more slowly. If they were like typical prokaryotes, with only one origin of replication per chromosome, DNA replication would take many days. 2.Instead, eukaryotic chromosomes contain multiple origins, at which DNA denatures and replication then proceeds bidirectionally until an adjacent replication fork is encountered. The DNA replicated from a single origin is called a replicon, or replication unit. 30

31 www.soran.edu.iq Chapter 3 slide 31

32 www.soran.edu.iq 3.In eukaryotes, replicon size is smaller than it is in prokaryotes, replication is slower, and each chromosome contains many replicons. Number and size of replicons vary with cell type. 4.Not all origins within a genome initiate DNA synthesis simultaneously. Cell-specific patterns of origin activation are observed, so that chromosomal regions are replicated in a predictable order in each cell cycle. 32

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34 www.soran.edu.iq Initiation of Replication 1.Eukaryotic origins are generally not well characterized; those of the yeast Saccharomyces cerevisiae are among the best understood. 2.Chromosomal DNA fragments (about 100bp) that are able to replicate autonomously when introduced into yeast as extracellular, circular DNA are known as ARSs (autonomously replicating sequences). 3.ARSs are yeast replicators. The three sequence elements typically found in ARSs are A, B1, and B2. 4.Initiator protein in yeasts is the multiprotein origin recognition complex (ORC), which binds to A and B1. Other replication proteins join, including one that unwinds DNA at B2. The yeast origin of replication is between regions B1 and B2. 5.DNA and histones must be doubled in each cell cycle. G1 prepares the cell for DNA replication, chromosome duplication occurs during S phase, G2 prepares for cell division, and segregation of progeny chromosomes occurs during M phase, allowing the cell to divide. 34

35 www.soran.edu.iq 6.Cell cycle control is complex, and only outlined here. Yeasts, in which chromosomal replication is well studied, serve as a eukaryotic model organism. 7.Initiation of replication has two separate steps, controlled by cyclin-dependent kinases (Cdks) that are present throughout the cell cycle, except during G1. a.In the absence of Cdsk during G1, replicator selection occurs. ORC and other proteins assemble on each replicator to from pre-replicative complexes (pre-RC). b.When cell enters S phase, Cdks are present, and activate pre- RCs to initiate replication. c.Cdk activity inhibits another round of pre-RC formation until the cell again enters G1, when Cdks are absent. 35

36 www.soran.edu.iq Fig. 3.x Some of the molecular events that control progression through the cell cycle in yeasts Some of the molecular events that control progression through the cell cycle 36

37 www.soran.edu.iq Eukaryotic Replication Enzymes 1. Enzymes of eukaryotic DNA replication aren’t as well characterized as their prokaryotic counterparts. The replication process is similar in both groups—DNA denatures, replication is semiconservative and semidiscontinuous and primers are required. 2.Fifteen DNA polymerases are known in mammalian cells: a.Three DNA polymerases are used to replicate nuclear DNA. Pol α (alpha) extends the 10-nt RNA primer by about 30nt. Pol δ(delta) and Pol ε(epsilon) extend the RNA/DNA primers, one on the leading strand and the other on the lagging (it is not clear which synthesizes which). b.Other DNA pols replicate mitochondrial or chloroplast DNA, or are used in DNA repair. 37

38 www.soran.edu.iq Replicating the Ends of Chromosomes 1. When the ends of chromosomes are replicated and the primers are removed from the 5’ ends, there is no adjacent DNA strand to serve as a primer, and so a single-stranded region is left at the 5’ end of the new strand. If the gap is not addressed, chromosomes would become shorter with each round of replication. 38

39 www.soran.edu.iq The problem of replicating completely a linear chromosome in eukaryotes 39

40 www.soran.edu.iq 2. Most eukaryotic chromosomes have short, species-specific sequences tandemly repeated at their telomeres. Blackburn and Greider have shown that chromosome lengths are maintained by telomerase, which adds telomere repeats without using the cell’s regular replication machinery. 3.In the ciliate Tetrahymena, the telomere repeat sequence is 5 TTGGGG-3. a.Telomerase, an enzyme containing both protein and RNA, binds to the terminal telomere repeat when it is single stranded, synthesizing a 3-nt sequence, TTG. b.The 3 end of the telomerase RNA contains the sequence AAC, which binds the TTG positioning telomerase to complete its synthesis of the TTGGGG telomere repeat. c.Additional rounds of telomerase activity lengthen the chromosome by adding telomere repeats. 40

41 www.soran.edu.iq 4.After telomerase adds telomere sequences, chromosomal replication proceeds in the usual way. Any shortening of the chromosome ends is compensated by the addition of the telomere repeats. 5.If the sequence of the telomerase RNA is mutated, telomeres will correspond to the mutant sequence, rather than the organism’s normal telomere sequence. Using an RNA template to make DNA, telomerase functions as a reverse transcriptase called TERT (telomerase reverse transcriptase). 6.Telomere length may vary, but organisms and cell types have characteristic telomere lengths. Mutants affecting telomere length have been identified, and data indicate that telomere length is genetically controlled. Shortening of telomeres eventually leads to cell death, and this may be a factor in the regulation of normal cell death. 41

42 www.soran.edu.iq Synthesis of telomeric DNA by telomerase 42

43 www.soran.edu.iq Assembling New DNA into Nucleosomes 1. When eukaryotic DNA is replicated, it complexes with histones. This requires synthesis of histone proteins and assembly of new nucleosomes. 2. Transcription of histone genes is initiated near the end of G1 phase, and translation of histone proteins occurs throughout S phase. 3.Assembly of newly replicated DNA into nucleosomes a.Parental histone cores separate into an H3-H4 tetramer, and two H2A-H2B dimers. b.H3-H4 tetramer (preexisting or newly made) binds to replicated dsDNA and begins nucleosome assembly. c.H2A-H2B dimers (preexisting or newly made) are added in an assembly process that requires histone chaperone proteins to direct it. 4.Self-assembly of nucleosomes has been observed only in vitro. 43

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