DNA Replication & Repair

1. DNA Replication & Repair

2. DNA Replication & Repair Dr. Ketki K Assistant Professor Dept of Biochemistry

3. The flow of information from DNA to RNA to protein is termed the …………………….

4. Structure of DNA • With the exception of a few viruses that contain single-stranded (ss) DNA, DNA exists as a double-stranded • 3′→5′-Phosphodiester bonds • by convention, always written in sequence from the …-end of the chain to the ….-end

6. Double helix • Chains are paired in an ………….fashion • Hydrophobic bases are stacked……….. • hydrophilic deoxyribose–phosphate backbone of each chain is on the………. • Base pairing :………… • Chargaff Rule :………..

8. When DNA is heated, the temperature at which one half of the helical structure is lost is defined as the melting temperature (Tm).

10. DNA Replication • During cell division, each daughter cell gets an exact copy of the genetic information of the mother cell, • This process of copying the DNA is known as DNA replication • In daughter cell, one strand is derived from mother cell while the other complementary strand is newly synthesized • This is c/a semiconservative type of DNA replication(one parent strand remains intact)

12. For easy understanding,replication is divided into • Initiation • Elongation • Termination

13. DNA replication in Prokaryotes-Initiation 1)In prokaryotic organisms, DNA replication begins at a single site called the origin of replication/ori composed almost exclusively of AT ( only 2 hydrogen bonds, so facilitates melting) 2) Two strands unwind & separate,creating replication fork 3)Replication fork move in both directions away from the origin generating replication bubble

16. DnaA protein binds specific nucleotide sequence( DnaA boxes/Replication recognition sequence),within the origin of replication • Causing AT rich region in ori to melt (ATP dependant process) • Results in strand separation with formation of SSDNA • DNA Helicase binds to SSDNA near replication fork, forcing strands apart (Energy requiring process, ATP)

17. DnaB is principle helicase in E coli ,requires DnaC also for binding to DNA • SSDBPs ( single stranded DNA binding proteins) binds to SSDNA and keeps two strands of DNA separated

19. Solving the problem of supercoils • Upon separation of two strands ,problem is encountered • Positive supercoils: in the region of DNA ahead of replication fork as a result of overwinding • Negative supercoils: in the region behind the fork • This problem interferes with further unwinding of DNA double helix

20. Type I DNA Topoisomerase: • strand cutting & strand resealing activity • Do not require ATP • Nick created in one strand→ intact DNA passed through the nick → resealed the nick → relaxing the accumulated supercoils • It relaxes negative supercoils in E coli, and both negative & positive supercoils in many prokaryotes and eukaryotes

21. Type II DNA Topoisomerase: • strand cutting & strand resealing activity • require ATP • Nick created in both strands→ second stretch of DNA passed through the nick → resealed the nick → relaxing the accumulated supercoils • It relaxes both negative & positive supercoils in prokaryotes and eukaryotes

22. Positive supercoiling resulting from DNA strand separation

23. Action of Type I DNA topoisomerases

25. Anticancer agent Camptothecins→ target human type I - topoisomerase Etoposide → target human type II- topoisomerase • Anti bacterial agent Ciprofloxacin (fluoroquinolones) → target bacterial DNA gyrase( type II topoisomerase)

26. DNA polymerase itself can not initiate synthesis of new strand, it requires RNA Primer(which contains RNA base paired to DNA template with free 3’-OH group) • 3’-OH group serves as first acceptor of deoxynucleotide by DNA Polymerase • RNA Primers are synthesized by Primase (specific RNA Polymerase,DnaG) at the replication fork.

28. Adding of primase converts proteins required for strand seperation into Primosomewhich makes RNA primers • Direction of DNA replication: DNA Pol III can synthesize new strands only in 5’ 3’ direction • and DNA replication on both strands should take place simultaneously

29. So, strand which is being copied in direction of advancing replication fork is c/a leading strand, whichis synthesized continuously • Strand which is being copied in direction away from replication fork is c/a lagging strand,whichis synthesized discontinuously • Lagging strand requires multiple RNA primers • With small stretches of DNA being copied near replication fork. these small stretches of discontinuous DNA on lagging strand, are termed as Okazaki fragments

31. Elongation

32. DNA Pol III elongates new DNA strand by adding deoxynucleotides to the 3’-end of growing chain (since 3’-OH group of RNA primer act as acceptor of first deoxynucleotide) • DNA Pol III is a processive enzyme ( it remains bound to template strand as it moves along,does not diffuse away and then rebinds before adding each new nucleotide) • Processivity is due to it’s β subunit forming a ring which encircles and move along template,serving as a sliding DNA clamp

33. With addition of each new nucleotide to the growing chain, PPi is released • Hydrolysis of PPi to 2 Pi gives energy to drive the reactions forward

35. For survival of organism,it is important to synthesize new strand with less no of errors, since misreading of template sequence can reduce deletorious lethal mutations

36. DNA Pol III has proof reading activity (3’ 5’ exonuclease), which removes mismatched nucleotides Example: C=A instead of C=G 3’ 5’ exonuclease removes mismatched nucleotide A 5’ 3’ polymerase activity replaces it with correct nucleotide containing G (excision must be in reverse direction from that of synthesis)

38. Excision of RNA primer and their replacement by DNA • DNA polymerase I : • has a 5′→3′ exonuclease activity that is able to hydrolytically remove the RNA primer • In addition it has 5′→3′ polymerase activity that synthesizes DNA, • and the 3′→5′ exonuclease activity that proofreads

39. First, 5′→3′ exonuclease activity of DNA Pol I is able to hydrolytically remove the RNA primer • Gap is created • Gap is filled by 5′→3′ polymerase activity of DNA Pol I that synthesizes DNA, • To remove the errors in newly synthesized strand, 3′→5′ exonuclease activity of DNA Pol I proofreads the new chain

41. This removal /synthesis /proofreading continues untill RNA primer is totally degraded,and gap is filled with DNA • Nick is present between two stretches of DNA in leading strand,between multiple stretches of DNA in lagging strand • Nick is sealed by DNA Ligase

42. DNA ligase • The final phosphodiester linkage between the 5′-phosphate group on the DNA chain synthesized by DNA polymerase III and the 3′-hydroxyl group on the chain made by DNA polymerase I is catalyzed by DNA ligase • This joining requires energy from hydrolysis of ATP to AMP and Ppi

45. Termination • Binding of protein TUS(terminus utilization substance) to replication termination site(ter sites) on the DNA, • Stopping the movement of DNA Polymerase

46. Termination • Mediated by sequence specific binding of protein,Tus(Terminus utilization substance) to replication termination site(ter sites) on DNA ,stopping the movement of DNA Polymerase

47. Eukaryotic DNA Replication

48. Eukaryotic cell cycle • Events of eukaryotic DNA replication & cell division are coordinated to produce cell cycle • G1 phase,S phase,G2 phase, M phase • Check points: cyclin & CDKs- it prevents entry into next phase of cycle untill preceeding phase has been completed