1. DNA replication Andy Howard Introductory Biochemistry 8 May 2008

2. 08 May 2008 DNA replication p.2 of 28 What we’ll discuss Prokaryotic DNA replication Semiconservative replication Unwinding of parent DNA Leading-strand replication Lagging-strand replication Okazaki fragments Eukaryotic replication Rates Multiple starting points Enzymes Other

3. 08 May 2008 DNA replication p.3 of 28 iClicker quiz 1. Which of these DNA sequences is palindromic? (a) TCGATG (b) TAGGAT (c ) GGCCCGGG (d) TACGCGTA (e) None of the above

4. 08 May 2008 DNA replication p.4 of 28 iClicker quiz #2 Suppose someone introduced a drug that interfered with deacylation of H1. What effect would this have on chromatin assembly? (a) It would prevent formation of the nucleosome core particle (b) It would interfere with assembly of the structures connecting one core particle to the next (c) Both (a) and (b) (d) It would have no effect on chromatin assembly (e) Like (c), except only in prokaryotes

5. 08 May 2008 DNA replication p.5 of 28 Semi-conservative replication A bit of a fanciful term Refers to the fact that, during DNA replication, each daughter molecule contains one of the strands of the parent Thus each daughter contains half (semi) of the original molecule This mode of inheritance was predicted by the Watson/Crick model Photo courtesy U. Costa Rica

6. 08 May 2008 DNA replication p.6 of 28 Meselson & Stahl 1958: showed that DNA really is replicated this way DNA grown with 15 N has higher density 15 N DNA allowed to replicate exactly once has intermediate density

7. 08 May 2008 DNA replication p.7 of 28 The E.coli chromosome One circular, double-stranded DNA molecule of about 4.6*10 6 bp Replication begins in only one place, i.e. a single origin of replication (OriC in E.Coli) Replication moves both directions until the two replication efforts meet at the termination site Protein machine that accomplishes replication is the replisome; one replisome in each direction Replication forks move 1000 bp/sec; thus E.coli can be replicated in 38 min

8. 08 May 2008 DNA replication p.8 of 28 By contrast: eukaryotes Bigger chromosomes, more of them Not circular, usually Fruit-fly chromosomes: Sex chromosome, 2 long autosomes, one tiny autosome 1.65 * 10 8 bp, 14000 genes Human 22 pairs of autosomes, sex chromosome 3.4*10 9 bp, ~22000 genes Replication is bidirectional as in E.coli More than one origin so replication is comparably fast even though rate is lower Drosophila chromosome TEM reconstruction

9. 08 May 2008 DNA replication p.9 of 28 How replication works in prokaryotes Takes place in the cytosol since there is no nucleus Specific enzymes form the molecular machine to carry out the task Has to involve separation of the strands Process divided into initiation, elongation, and termination Enzymatic functions identified for each segment

10. 08 May 2008 DNA replication p.10 of 28 Prokaryotic DNA polymerases Several varieties DNA polymerase III is the one responsible for most of the work (but the 3rd discovered); it’s the biggest and most complex DNA pol I involved in error correction and helps with replication of one of the strands DNA pol II also does DNA repair Multi-subunit, complex entities

11. 08 May 2008 DNA replication p.11 of 28 DNA Pol III Diagram from Kelman et al (1998) EMBO J. 17:2436

12. 08 May 2008 DNA replication p.12 of 28 Components of DNA Pol III SubunitMr,kDaGeneActivity  130polC/dnaEPolymerase  27dnaQ/mutD3’-5’ exonuclease  8.9holEStabilizes proofreading by  ?  40dnaNSliding clamp  71dnaXDimer, ATPase  complex 15-47VariousProcessivity

13. 08 May 2008 DNA replication p.13 of 28 So how does it work? Add 1 nucleotide @ a time to 3’ end of growing chain Substrate is a dNTP Watson-Crick bp determines specificity Enzyme spends 75% of time tossing out wrong bases Forms phosphodiester linkage Pol III remains bound to the replication fork Diagram from answers.com

14. 08 May 2008 DNA replication p.14 of 28 Error correction in DNA pol III 3’-5’ proofreading recognizes incorrectly paired bases and repairs most of them This is an exonuclease activity because it clips off the last nucleotide in the chain 10 -5 inherent error rate drops to 10 -7 because the exonuclease goofs 1% of the time Separate repair enzymes drop that down to 10 -9

15. 08 May 2008 DNA replication p.15 of 28 Processivity This term refers to the fact that many nucleotides can be added to a growing chain following a single association event in which the polymerase (e.g. E.coli Pol III) associates with the template DNA. We describe replication as highly processive if 50,000 bases can be replicated based on a single association of Pol III with our template.  subunits slide along, which is how this is done  complex is responsible for keeping the polymerase attached so that this is possible

16. 08 May 2008 DNA replication p.16 of 28 Initiation Begins in E.coli at a single origin called OriC DnaA binds to origin—region called DnaA box Replication fork forms after it binds Helicases & primases set up for starting replication Complementary RNA tag attached at the replication fork

17. 08 May 2008 DNA replication p.17 of 28 Elongation DNA polymerase operates in 5’-3’ direction on both strands For one strand that’s straightforward replication moves in direction of unwinding of the DNA This is known as leading-strand synthesis For the other strand it must work opposite to the unwinding therefore it’s more complicated This is lagging-strand synthesis

18. 08 May 2008 DNA replication p.18 of 28 Termination Replication needs to know how to stop Defined sequence ter is opposite the origin on the chromosome Specific enzyme, Tus, involved in recognizing termination signals Ter has sequences that play a role in separating the daughter chromosomes Tus-Ter complex; images courtesy Memorial Univ., Newfoundland

19. 08 May 2008 DNA replication p.19 of 28 Leading-strand synthesis One base at a time is incorporated by  subunit of DNA polymerase, complementary to existing strand At some point RNA primer is replaced with DNA Image courtesy U.Pittsburgh

20. 08 May 2008 DNA replication p.20 of 28 Lagging-strand synthesis Movement of enzyme is opposite to unwinding Therefore it must work a few bases (~1000) at a time and then back up The segments thus formed on the lagging strand are known as Okazaki fragments DNA Pol I removes RNA primer DNA ligases link together Okazaki fragments

21. 08 May 2008 DNA replication p.21 of 28 Primases These are DNA-dependent RNA polymerase enzymes that initiate DNA synthesis, particularly on the lagging strand, where you need to do that at the beginning of each Okazaki fragment DnaG helicase binding domain PDB 2R6A 347 kDa trimer of heterotrimers Bacillus stearothermophilus

22. 08 May 2008 DNA replication p.22 of 28 Role of DNA Pol I Part of the system for producing a continuous DNA strand on the lagging side Contains both 5’  3’ polymerase activity and 3’  5’ proofreading exonuclease activity Also has 5’  3’ exonuclease activity: that’s used to remove the RNA primer

23. 08 May 2008 DNA replication p.23 of 28 Rates and sequencing Because there’s only one place where replication can begin, the process has to occur in discrete steps The enzymes themselves are efficient, because they move with the unwinding of the double helix Typical rates 1000 nucleotides/ sec So for E.coli it takes 38 min = 2280 sec to replicate the entire chromosome (4.6*10 6 bp) / [(10 3 bp/sec)(2 directions)]= 2300 sec

24. 08 May 2008 DNA replication p.24 of 28 Where are things happening? Both leading- and lagging-strand synthesis are catalyzed in both the clockwise and counterclockwise directions. Each DNA Pol III molecule is catalyzing both leading- and lagging-strand synthesis.

25. 08 May 2008 DNA replication p.25 of 28 Eukaryotic DNA polymerases More complex, as you’d expect More than one initiation point Therefore even though the enzymes work less rapidly, they can multiplex the process The result is that human DNA can be replicated in roughly the same time scale as E.coli DNA image courtesy Memorial Univ., Newfoundland

26. 08 May 2008 DNA replication p.26 of 28 Eukaryotic DNA polymerases Several complexes involved:  Elongation and repair  Elongation and repair  Elongation and repair  DNA repair  Helps in replicating mitochondrial DNA chloroplast polymerase Don’t fall into the trap: these Greek letters don’t necessarily mean the same thing that they mean in the context of bacterial replication! Human DNA polymerase  : From answers.com

27. 08 May 2008 DNA replication p.27 of 28 Specific roles for  and   complex does leading-strand synthesis and 3’-5’ exonuclease activity, which is impressively effective  and  involved in lagging strand synthesis:  is DNA polymerase and RNA primase;  extends segment to complete Okazaki fragment

28. 08 May 2008 DNA replication p.28 of 28 Roles for DNA polymerase  Big,multi-subunit protein Similar to DNA pol I from E.coli It repairs and it fills gaps between Okazaki fragments Largest piece is a polymerase and does 3’  5’ proofreading Cryo EM image from Asturias et al (2006) Nature Struct Mol Biol 13:35 ; yeast