Prokaryotic DNA Replication

Transcription

1 Prokaryotic DNA Replication DNA replication is perfomed by a multienzyme complex >1 MDa DNA Nucleotides Replisome: DNA polymerases Helicase Primase SSBs DNA ligase Clamps (Topoisomerases) 1

2 Replication is semiconservative, accurrate and fast Accuracy 1 error in 1 billion bases Speed 500 nt/s in bacteria 50 nt/s in mammals Each original strand functions as template for DNA synthesis 2

3 After each replication cycle, DNA is doubled DNA is synthesized in 5 to 3 direction 3

4 Polymerisation in detail (dnmp) n + dntp (dntp) n+1 + PP i DNA 2 P i Complementary basepairing and matching hydrogen bonds is required Incorrect basepairing 4

5 DNA is synthesized by DNA polymerase DNA polymerase III is a protein complex Subunit function not known 3 exonuclease polymerase clamp dimerisation clamp loader 5

6 E. coli contains multiple DNA polymerases DNA pol I DNA pol II DNA pol III Number/cell Speed (nt/s) exonuclease Yes Yes No 5 exonuclease Yes No No Processivity Role DNA repair RNA primer removal DNA repair Replication DNA polymerase I Found by Arthur Kornberg, mid 1950 s Three enzymatic activities: Polymerase activity 3 to 5 exonuclease activity 5 to 3 exonuclease activity Klenow enzyme is lacking one subunit responsible for the 5 to 3 exonuclease activity 6

7 DNA polymerase requires 1. A free 3 -OH group supplied by RNA Primer for start of polymerisation 2. Mg 2+ ions for activity in active site 3. A template to copy DNA replication initate at origin of replication Bacterial chromosome doubles in 40 min 7

8 DNA replication is bidirectional The replication origin OriC in E.coli 245 base pairs AT-rich Initiation proteins bind to 9 bp consensus sequence 8

9 Inititation of replication at the replication origin Regulation of initiation of replication 9

10 DNA is synthesized in the replication fork in 5 to 3 direction Leading strand synthesis is continuous whereas lagging strand is synthesized in fragments Length of Okazaki fragments in prokaryotes are nt, in eukaryotes nt 10

11 Mistakes during DNA synthesis are edited This results in a very low error rate of 1 in 1 billion nucleotides 3 to 5 exonuclease activity corrects errors 11

12 Requirements for proofreading mechanism Addition of nucleotides to RNA primer Absolute requirement for a match at the 3 end of the extended strand 3 to 5 exonuclease activity of DNA polymerase Template DNA is identified by methylation (E. coli) or absence of nicks (eukaryotes) 5 to 3 exonuclease activity causes strand displacement/nick translation No net synthesis 12

13 Helicase unzips double-helix Single strand binding proteins keep strands single stranded Each SSB bind to 7-10 nt Bind in clusters Cooperative binding Lowers Tm of template 13

14 Binding of SSBs to DNA DNA pol. is attached to strand by Clamp loader and Sliding clamp 14

15 Sliding clamp Accounts for high processivity: Limits association and dissociation 15

16 DNA primase Makes the 10 nt RNA primer required for start of replication In beginning of each Okazaki- Fragment RNA primer is later erased and replaced with DNA by DNA pol I 16

17 DNA ligase Seals the nicks between Okazaki fragments Requires close and free 3 -OH and 5 -P and proper base-pairing NAD+ required in prokaryotes ATP required in eukaryotes Nick sealing by DNA ligase 17

18 Topoisomerases Relieves torsional stress caused by rotation of DNA ahead of the fork 10 nucleotides = 1 turn Topoisomerase I Breaks one strand of the duplex 18

19 Mechanism of topoisomerase I 19

20 Topoisomerase II (DNA gyrase) Breaks both strands of the duplex Introduces negative superhelices ATP dependent 20

21 Summary of replication DNA is bent duing replication process 21

22 DNA is proofread during the process Termination of replication The two replication forks are synchronized by bp Ter sequences that bind Tus proteins Tus proteins can only be displaced by replisomes coming from one direction 22

23 Resolvation of replication products by decatenation Eukaryotic DNA Replication 23

24 Eukaryotes has some special features Larger genome Multiple linear chromosomes Centromers Telomeres Histones DNA replication DNA replication takes place during the S phase part of the interphase of the cell cycle. S for synthesis. Two identical copies of the chromosome are produced, attached at the centromer. 24

25 Parts on the yeast chromosome contain Autonomous Replicating Sequence Eukaryotes also contain multiple DNA polymerases DNA pol DNA pol DNA pol DNA pol DNA pol 3 exonuclease No No Yes Yes Yes Fidelity x Processivity Moderate Low High High High Role Lagging strand primer synthesis DNA repair Mitochondria l DNA replication Lagging strand replication Leading strand replication 25

26 Inititiation of replication in eukaryotes Due to the eukaryotic chromosome size, multiple replication origins are needed Eukaryotic replication origins are organized in replicons, ori/cluster Replication is initated all through the S phase Active chromatin replicate early, condensed chromatin replicate late A replication bubble is formed at each ori, forks moving in both directions Each ori is only replicated once Histones are synthesized only during S phase and are added as replication proceeds Some histone parts are inherited some are new The spacing of histones every 200 nt might be the reason for the shorter Okazakifragments in eukaryotes and the slower speed of replication 26

27 New histones are modified Telomerase recognizes the G-rich 3 - end of the chromosome (telomere) 27

28 Comparison prokaryotic vs eukaryotic replication Prokaryote (E.coli) Eukaryote (Human) # Origins of replication in replicons Speed of replication 500 nt/s 50 nt/s Time for replication 40 min 8 hours Okazaki fragments nt nt Polymerases 3 (5) 5 (10) Chromosomes 1, circular 46, linear Other Telomeres, histones 28

29 Reverse transcription Retroviruses are mobile genetic elements 29

30 RNA-dependent DNA polymerase 30

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