DNA replication

1. DNA replication 醫技系 楊孔嘉

2. References 1. Nature 2003, 421:p431 (replication & recombination) 2. EMBO reports 2003, 4:p666 (replication) 3. BioEssays 2003, 25:p116 (MCM proteins) 4. Gene 2003, 310:p1 (ORC cycle) 5. FEMS Microbiol Rev 2003, 26:p533 (structure of OBPs) 6. Cancer Lett 2003, 194:p139 (telomerase) 7. J Struct Biol 2003, 140:p17 (initiation) 8. EMBO reports 2003, 4:p1 (kinase) 9. Curr Opin Microbiol 2003, 6:p146 (cell size) 10. Genome Biol 2003, 4:p204 (microarray) 11. Annu Rev Biochem 2002, 71:p333 (eukaryotes) 12.Nature Cell Biology 2004, 6:p648 (origin firing) 13. Acta Biochimica Polonica 2005, 52:p1 (SSB) 14. GENES VIII chapter 14 (Introduction)

4. Learning objectives Let the students know where we are and where to go about DNA replication. Prokaryotic cells Eukaryotic cells the same or different ?

5. Molecular structure of nucleic acid “This structure has novel features which are of considerable biological interest ” Nature 171, April 25 (1953) p. 737-738 - by James Watson and Francis Crick

6. The principles of DNA replication • template dependent • requires the four dNTPs • primer-dependent • synthesizes DNA in the 5' to 3' direction, reading the template 3' to 5'

7. The principles of DNA replication

9. The central questions to be answered • Does DNA synthesis begin at a defined place ? (2) What determines replication initiation sites ? (3) What regulates an origin to fire once and only once per cell cycle ?

11. Chronicle for DNA replication 1950 1960 1970 1980 1990 2000 In 1972, Kornberg et al. demonstrated in vitro enzymatic activity of DNA polymerase from E. Coli J. Biol. Chem. 1972;247:p241.

12. Chronicle for DNA replication In 1984, the first mammalian-based DNA replication system (SV40) that initiated DNA synthesis successfully in vitro was developed. Proc Natl Acad Sci U S A. 1984;81:p6973. 1950 1960 1970 1980 1990 2000 In 1985, Greider and Blackburn identified enzymatic activity of the telomerase in Tetrahymena extract Cell 1985;43:p405.

13. Chronicle for DNA replication Establish the molecular mechanism of DNA replication, involving the stages of initiation, priming, elongation and termination 1950 1960 1970 1980 1990 2000 Establish the linkage of DNA replication to cell cycle control

14. E. Coli model • Yeast genetic model • Simian virus 40 (SV40) model • Adenovirus model • Herpes simplex virus (HSV) model Study on DNA replication Genetic approach: temperature sensitive(ts) mutants Biochemical approach: in vitro complementation assay

15. The principles of DNA replication • template dependent • requires the four dNTPs • primer-dependent • synthesizes DNA in the 5' to 3' direction, reading the template 3' to 5'

16. Priming for DNA replication • DNA polymerases is incapable of initiating DNApolymerization in the absence of an existing 3' -OH • The priming RNA supplies the necessary 3' -OH group to primeDNA polymerization and is later clipped off and replaced with DNA by the DNA polymerase

17. Priming for DNA replication • Due to low fidelity of RNA polymerases, primase and primosome takes over for priming RNA synthesis. • Okazaki fragment formation requires repeated initiation of DNA replicationde novo. • Three kind of priming: an RNA primer, a nick in DNA, a priming protein

18. Priming for DNA replication • RNA primer: The majority of prokaryotic and eukaryotic DNA replication • DNA primer: nick translation, hairpin loop • Protein (terminal protein + dCTP) as a primer: adenovirus DNA replication

19. The principles of DNA replication • template dependent • requires the four dNTPs • primer-dependent • synthesizes DNA in the 5' to 3' direction, reading the template 3' to 5'

20. Why DNA synthesis is 5’ to 3’ ? energy source energy source

21. The principles of DNA replication • Replication complex and replication fork • Bi-directional DNA replication • Enzymology of DNA replication

22. DNA replication fork The DNA replication fork Leading strand Lagging strand with Okazaki fragments (1000-2000 base) Most recently synthesized DNA

23. The principles of DNA replication • Replication complex and replication fork • Bi-directional DNA replication • Enzymology of DNA replication

24. Directions of DNA replication Directions of DNA replication

25. Experimental evidence of bidirecitonal DNA replication

26. Large eukaryotic DNA has multiple origins of replication

27. The principles of DNA replication • Replication complex and replication fork • Why DNA synthesis is 5’ to 3’ • Bi-directional DNA replication • Enzymology of DNA replication polymerase, primase, helicase, single strand DNA binding protein (SSB), ligase, topoisomerase, telomerase

28. Prokaryotic system

29. DNA polymerase of E. coli Proofreading function *Polymerases IV and V participate in SOS repair function.

30. E. Coli DNA Pol III (replicative polymerase) • complex enzyme with ten subunits • a subunit contains the active site for nucleotide addition • e subunit is a 3’ to 5’ exonuclease, a proofreading activity that removes incorrectly added bases • q subunit can stimulate exonuclease

31. E. Coli DNA Pol III (replicative polymerase) • b subunit is a processivity factor -> prevents enzyme from falling off prematurely thus decrease likelihood of frameshift mutation • g subunit is a clamp loader -> places the processivity subunit on DNA • t subunit is a dimerizing subunit -> link the two catalytic cores together Note: In mammalian system: PCNA is a processivity factor, RF-C is a clamp loader

32. 3D structure of DNA polymerase PDB: 1D8Y Rotate 60 FingerThumb nucleotide Palm DNA 3‘-5’exonuclease

33. 3D structure of DNA polymerase Editing mode Polymerizing mode

34. b-subunit clamp bsubunit water water DNA bsubunit

35. Assembly of E. Coli DNA Pol III Clamp loader (g) cleaves ATP to load clamp (b) on DNA Enter the core enzyme (a, e, q)

36. Assembly of E. Coli DNA Pol III Repetitively loading on the lagging strand Dimerization by t subunits

37. Stages of DNA replication • Initiation • Elongation • Termination • Note: At the initiation stage, origin binding proteins (OBP) • and ATP is required to trigger the following events • Melt the two strands of DNA • Recruit protein factors essential for replication

38. Origin for DNA replication in E. coli 245 bp ori C region 13 bp sequencesDnaA-binding sites (3 repeats) (4 repeats of 9 bp) • Seven replication factors working at the origin • DnaA: origin binding protein (OBP) • DnaB: helicase • DnaC: recognition protein • HU: bend DNA structure • Gyrase: rotate one strand over the other • SSB: single strand binding • DnaG: primase

39. 3D structure of DnaA ATPases Associated with various cellular Activities (AAA+) DBD Mg2+ ADP cofactor Note: Common features are shared in the initiator of chromosomal replication in bacteria, archaea and eukaryotes FEMS Microbiology Review 2003, 26, p533

40. Initiation of DNA replication in E. coli A-T rich region DnaG DnaB DnaB DnaG DnaA DnaB DnaB

41. Origin for DNA replication in E. coli Prepriming complex Replication bubble DnaG DnaB DnaB DnaG DnaA DnaB DnaB

42. Regulatory inhibition of DnaA DnaG DnaB DnaB DnaG DnaB DnaB Dna A DnaA protein undergoes conformation change in hydrolysis of ATP and unable to initiate further replication rounds Note: DnaA protein can be reactivated either by acidic phospholipids or DnaK chaperon, that exchange ADP by ATP

43. Competition model for DnaA-ATP and DnaA-ADP:coupling the initiation of chromosome replication to cell size Current Opinion in Microbiology 2003, 6:p146 Total DnaA-ADP (inactive) Total DnaA-ATP (active) Cell growth New DnaA-ATP (active) Old DnaA-ATP (active) eclipse DNA replication completion of cell division

44. The factors that regulate an origin to fire only once per cell cycle • Convert DnaA-ATP to DnaA-ADP • Repress DnaA transcription • Prevent DnaA binding -> DNA replication ->Hemimethylated GATC at the oriC DNA -> sequestered to plasma membrane -> thus a membrane-bound inhibitor prevents re-initiation of the origin by DnaA

45. DNA helicase DNA helicase ~ 60 bp

46. Single strand binding protein (SSB)

47. Single strand binding protein (SSB) • The SSBs from prokaryotes and eukaryotes share a common core ssDNA-binding domain • Application of SSB in biotechnology • Increase amplification efficiency in single PCR • Prevention of primer dimer formation in multiplex PCR • Increased size of cDNA in RT-PCR

48. Replication complex and replication fork Dimer of Pol III Primase Leading strand Okazaki fragment Leading strand template Lagging strand template Single strand binding protein (SSB)

50. Lagging strand synthesis in E. Coli