DNA Recombination and Repair Wenya Huang Medical Laboratory Science and Biotechnology

1. DNA Recombination and Repair Wenya Huang Medical Laboratory Science and Biotechnology National Cheng-Kung University

2. Outline • DNA recombination • DNA damage • DNA repair • Assays for DNA damage and repair

3. 3 ‘R’s of DNA metabolism: Replication (copying of DNA prior to each cell division) Recombination (exchanges between different DNA molecules in a cell) Repair (restoration of altered DNA to its normal state)

4. recombination

5. Recombination in circular DNA attP attB e.g., phage integration into bacterial genome Cited from Genes VIII

6. Homologous Recombination • General (meiosis): chromosomal exchange between genes on the same locus. It occurs at the “four-strand stage of meiosis. • 2. Specialized (site-specific) recombination: recombination between specific pairs of sequences. It is common in prokaryotes. E.g., phage DNA integration into bacteria, which include a short stretch of homology. • 3. Transposition by transposons

7. Homologous recombination - Guided by base-pairing interactions between two homologous DNA molecules • Two homologous DNA molecules in different chromosomes “cross over”.Their double helices break and the two broken ends join to their opposite partners to re-form two intact double helices, each composed of parts of the two initial DNA molecules. • The site of exchange can occur anywhere in the homologous nucleotide sequences of the two participating DNA molecules.

8. At the site of exchange, a strand of one DNA molecule becomes base-paired to a strand of the second DNA molecule to create a heteroduplex joint that links the two double helices. This heteroduplex joint can be thousands of base pairs long. • No nucleotide sequences are altered at the site of exchange. The cleavage and rejoining events occur so precisely that not a single nucleotide is lost or gained.

9. Meiotic recombination – initiated by double-strand DNA breaks • The process begins when an endonuclease makes a double-strand break in a chromosome. An exonuclease then creates two protruding 3’ single-stranded ends, which find the homologous region of a second chromosome to begin DNA synapsis. The joint molecule formed can eventually be resolved by selective strand cuts to produce two chromosomes that have crossed over.

10. Recombination in meiosis Cited from Genes VIII

11. DNA synapsis At DNA synapsis, the base pairs form between complementary strands from the two DNA molecules. This base-pairing is then extended to guide the general recombination process, allowing it to occur only between DNA molecules that contain long regions of matching DNA sequence.

12. Homologous recombination guided by DNA double-strand break Cited from Mol Biol. Cell, 4th edition

13. Recombination • Double-strand break • Unwinding and trimming • Strand invasion (single-strand assimilation) • Strand migration and displacement • DNA synthesis • resolution Cited from Genes VIII Cited from Genes VIII

14. Recombination In E. coli: RecABCD system • Activate RecBCD nuclease and helicase • RecBCD nuclease and helicase • - RecA • -DNA polymerase • Branch migration (RecA and RuvAB) • DNA polymerase Cited from Genes VIII

15. Recombination in E. coli: RecABCD Nuclease and helicase Cited from Genes VIII

16. RecBCD binds to DNA at a double-strandedend. Two of its subunits have helicase activities: RecD functions with 5’-3’ polarity, and RecB functions with 3’-5’ polarity. • RecD initially translocates along DNA and unwinds the double helix. • RecBCD degrades the released single strand with the 3’ end. When it reaches the chi site, it recognizes the strand of the chi site in single-stranded form. It cleaves the strand of the DNA at a position between four and six bases to the right of chi. • Recognition of chi site causes the RecD subunit to dissociate or become inactivated, the nuclease activity is then lost. • RecBC cointinues to perform the helicase activity.

17. Chi sequence 5’ GCTGGTGG 3’ 3’ CGACCACC 5’ Occur naturally in E. coli DNA per 5-10 kb Stimulate recombination in its vicinity (< 10 kb) Targets for the RecBCD complex

18. RecA (in E. coli) • Binds to single-strand DNA to form a nucleoprotein filament. • It is also a DNA-dependent ATPase. • It associates much more tightly with DNA when it has ATP bound. • Each RecA monomer has more than one DNA-binding site to hold a single strand and a double helix together. It catalyzes a multi-step DNA synapsis reaction between a DNA double helix and a homologous region of single-stranded DNA. It helps formation of the three-stranded intermediate. • It enables a DNA single strand to pair with a homologous region of DNA double helix. • It catalyzes unidirectional branch migration, readily producing a region of heteroduplex DNA that is thousands of base pairs long.

19. RecA protein and DNA synapsis

20. RecA: Invading of DNA with free end Cited from Genes VIII

21. RecA homologs in eukaryotes • There are at least 7 RecA homologs in humans and mice. These include Rad51, Rad50, Rad52, BRAC1 and BRAC2, etc. • Each homolog has its special catalytic activities and its own set of accessory proteins. • Among these proteins, Rad51 plays the central roles. It is functionally most similar to RecA.

22. Branch migration After DNA synapsis has occurred, an unpaired region of one of the single strands displaces a paired region of the other single strand, moving the branch point without changing the total number of DNA base pairs.

23. Branch migration Cited from Genes VIII

24. Branch migration Cited from Genes VIII

25. Heteroduplex

26. RuvAB complex: branch migration Cited from Genes VIII

27. RuvAB complex • It processes the Holliday junctions into mature recombination products • RuvA binds the Holliday junctions as a tetramer or double tetramer with high affinity and unfolds the junctions from the stacked X-structure. • RuvB targets the Holliday junction by interaction with the RuvA-junction intermediate. Two hexameric rings of RuvB encircle opposing DNA duplex arms of the junction and act as ATP-dependent DNA motors extruding heteroduplex DNA.

28. Holliday junction • In a Holliday junction, the two homologous DNA helices that have initially paired are held together by the reciprocal exchange of two of the four strands present, one originating from each of the helices.

29. Crossing over: Holliday junction

30. Holliday junction

31. Resolution of Holliday junction Cited from Genes VIII

32. RuvABC in bacteria Cited from Genes VIII

33. RuvC • It is also called DNA resolvase • It is a Holliday junction-specific endonuclease, which cleaves Holliday junction into two mature recombinant products

34. Homologous and non-homologous recombination pathways for double-stranded DNA breaks Cited from Mol Biol. Cell, 4th edition

35. Homologous recombination (HR) takes place in M (meiosis) and late S–G2 (DNA repair) phases and involves the generation of a single-stranded region of DNA, followed by strand invasion, formation of a Holliday junction, DNA synthesis using the intact strand as a template, branch migration and resolution.

36. Non-homologous end-joining (NHEJ) takes place throughout the cell cycle and involves binding of the KU heterodimer to double-stranded DNA ends, recruitment of DNA-PKcs (officially known as protein kinase, DNA-activated, catalytic polypeptide (PRKDC)), processing of ends (including Artemis-dependent processing) and recruitment of the DNA ligase IV (LIG4)–XRCC4 complex, which brings about ligation.

37. Mammalian

39. Ku70, Ku80 and DNA strand ends Cited from Genes VIII

40. Mre112Rad502Xrs2(Nbs1) pentamer • It is named MRN (mammals) or MRX (yeast). • The terminal CXXC-hooks form an interlocking dimerization interface in the Rad50 coiled-coils. • Intermolecular dimerization between individual complexes allows MRX (MRN) complex to tether DNA ends and perhaps tether and align sister chromatids.

42. Rad50 • catalytic domain: intramolecular ATP-binding cassette (ABC) type ATPase domain. Rad50 forms homodimers in the presence of ATP. Dimerization creates a DNA-binding interface across the Rad50 dimer. • Mre11 binds to the base of the coiled-coil near the Rad50 DNA-binding interface, suggesting the formation of a composite DNA-binding site within the (Mre11)2/(Rad50) 2 heterotetramer.

43. Mre11 nuclease • 3'-5' exonuclease activity on blunt and 3' recessed ends. • endonuclease activity on circular and linear ssDNA. • endonuclease cleavage of hairpin ends and 3' ssDNA overhangs at the single-/double-stranded transition. • Rad50 and Xrs2 (Nbs1) both influence MRX substrate binding and potentiate the intrinsic nuclease activity of Mre11

44. The MRE11/RAD50/NBS1 complex It is the central player in the cellular response to DNA double-strand breaks, including homologous recombination, non-homologous end joining and telomere breakage. The Mre11 complex possesses an ATP-stimulated nuclease to process heterogeneous DNA ends and long coiled-coil tails to link DNA ends and/or sister chromatids.

45. Xrs2 (yeast) or NBS1 (mammals) Nbs1 directly interacts with Mre11. The MRN complex plays critical roles in initiation of the intra S-phase checkpoint in response to DNA damage, and Nbs1 is phosphorylated as part of the damage signal by kinases from the ATM family of protein kinases.

46. Functional or physical interactions of the MRN complex with other DNA repair/checkpoint proteins

47. DNA replication, transcription and recombination cause topological changes in DNA structures.

48. DNA replication and topology 1. 2. Cited from Genes VIII

49. Resolution of topological changes of DNA molecules Cited from Genes VIII

50. DNA Topoisomerase • Relax or introduce supercoils in DNA • Transiently break in one or both strands of DNA, passing the unbroken strands through the gap and then resealing the gap.