DNA Recombination Mechanisms

1. DNA Recombination Mechanisms AHMP 5405

2. Objectives • List the major classes of mobile genetic elements (we went over this before) • Describe the process of general recombination • Diagram the process of gene conversion via Holliday junctions • Describe ways by which site-specific recombination can influence DNA rearrangement and genetic regulation

3. Recombination repair • Present in prokaryotic and eukaryotic cells • Only poorly understood • We know it exists because UvrA- and RecA- cells are much more sensitive to UV than cells containing only one mutation

4. Why do chromosomes undergo recombination? • Deleterious mutations would accumulate in each chromosome • Recombination generates genetic diversity

5. Recombination ABCDEFGHIJKLMNOPQRSTUVWXYZ abcdefghijklmnopqrstuvwxyz ABCDEFGhijklmnoPQRSTUVWXYZ abcdefgHIJKLMNOpqrstuvwxyz

7. Mitotic and meiotic recombination • Recombination can occur both during mitosis and meiosis • Only meiotic recombination serves the important role of reassorting genes • Mitotic recombination may be important for repair of mutations in one of a pair of sister chromatids

8. Recombination mechanisms • Best studied in yeast, bacteria and phage • Recombination is mediated by the breakage and joining of DNA strands

9. The Holliday model • Two homologous duplexes are aligned • Strand exchange leads to an intermediate with crossed strands • This branch can move: Branch migration • The branch is resolved by cleavage and sealing

10. Double strand break model

11. Double strand break model

12. Double-strand breaks in DNA initiate recombination (part I)

13. Double-strand breaks in DNA initiate recombination (part II)

14. The cross-strand Holliday structure is an intermediate in recombination (part I)

15. The cross-strand Holliday structure is an intermediate in recombination (part II)

16. Initiation of recombination by the RecBCD enzyme

17. Branch migration and resolution of Holliday structures depends on Ruv proteins

18. Action of E. coli proteins in branch migration and resolution of Holliday structures

19. Chi structures • When plasmids recombine figure eight structure is formed • If the recombined plasmids are cut with a restriction enzyme a c (chi) is formed

20. Generation of a chi intermediate

21. Electron micrograph of the chi form

22. What does the Chi structure prove? • The fact that each pair of arms is the same length shows that the circles are joined at homologous sites

23. Recombination between homologous DNA sites • Recombination provides a means by which a genome can change to generate new combinations of genes • Homologous recombination allows for the exchange of blocks of genes between homologous chromosomes and thereby is a mechanism for generating genetic diversity • Recombination occurs randomly between two homologous sequences and the frequency of recombination between two sites is proportional to the distance between the sites

24. Cre protein and other recombinases catalyze site-specific recombination

25. The mechanism of Cre-loxP site-specific recombination

26. Site specific recombination • Viruses and transposable elements often integrate their genomes into the host chromosome • Site specific recombination is used by both eukaryotes and prokaryotes to regulate gene expression and to increase the organisms genetic repertoire

27. Site specific recombination

28. Mechanism of gene rearrangement

29. V(D)J recombination

30. Repair by End Joining (Recombination Repair)

31. DNA non-homologous end-joining (NHEJ) • Predominant mechanism for DSB repair in mammals. • Also exists in single-celled eukaryotes, e.g. Saccharomyces cerevisiae • Particularly important in G0/G1

32. DNA-PKcs Homologous recombination Non-homologous end-joining DSB DSB Rad50, Mre11, Xrs2 complex Resection Ku70, Ku80 Rad52 Rad50, Mre11, Xrs2 complex Strand invasion “Cleaning up” of ends Rad51; BRCA2 DNA synthesis     XRCC4/Ligase IV Ligation Ligation, branch migration, Holliday junction resolution

33. DNA-PK DNA-PK DNA-dependent protein kinase (DNA-PK) DNA ACTIVE KINASE INACTIVE

34. K u 7 0 K u 8 0 DNA-PK has three subunits D N A X P D N A - P K c s K u 7 0 K u 8 0 6 9 k D a D N A - P K c s 8 3 k D a A T P A D P 4 7 0 k D a A C T I V E I N A C T I V E Target sites: Ser/Thr-Gln

35. K u 7 0 K u 8 0 DNA-PK has three subunits D N A X P D N A - P K c s K u 7 0 K u 8 0 6 9 k D a D N A - P K c s 8 3 k D a A T P A D P 4 7 0 k D a A C T I V E I N A C T I V E … and is activated by DNA DSBs!

36. Multiple potential roles for Ku/DNA-PKcs in NHEJ

37. End-joining repair of nonhomologous DNA

38. NHEJ: links to cancer • Status of NHEJ helps to define clinical radiosensitivity: • Defects in DNA ligase IV associated with cells of a radiosensitive leukaemia patient (180-BR). • Levels DNA-PK correlate with clinical outcome (cervical cancer). • Inherited or somatic defects in DNA-PK system may lead to cancer.

39. ATM: deficient in ataxia-telangiectasia (A-T) • Human autosomal recessive disorder • Progressive neurodegeneration • Cancer predisposition • Aspects of premature ageing • Radiosensitivity • Impairment in triggering cell cycle checkpoints in response to DNA DSBs

40. Rad24 Mec3 Ddc1 Rad9 Rad17 P P Tel1 Mec1 P P Chk1 Rad53 FHA FHA P Dun1 FHA Cdc25 DNA-damage signalling is conserved from yeast to man S. cerevisiae H. sapiens S. pombe P Rad17 Rad17 Hus1 Hus1 Rad9 Rad9 Crb2 Brca1? Rad1 Rad1 P P P P ? Lcd1 Rad26 Tel1 ATM ATR Rad3 P p53 P P P P P Chk1 Chk1 Cds1 Chk2 FHA FHA Cdc25C Cdc2 activation Pds1 destruction Cdc5 Cdc2 (Cdk1) G1-S Esp1 activation Crt1, Sml1? activation