1. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E NHEJ Cleaning up loose ends...

2. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

3. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E VDJ Joining: an overview Unique recombination signal sequences: RSS’s 3’ to each V gene segment 5’ to each J gene segment Flanking each D gene segment Each has –conserved palindromic heptamer –conserved AT-rich nonamer –spacer of 12 (one turn) or 23 (two turn) bases One turn RSS can only join with two turn RSSs

4. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E VDJ Joining: an overview Recombination and Orientation –Joining can result in deletion of the signal joint and intervening DNA inversion of the DNA –Determined by orientation of genes/RSS’s –Lymphoid specific requirements: RAG-1, RAG-2, Tdt –All other factors (many others) not lymphoid specific

5. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Stepwise Mechanism Recognition of RSSs and Synapsis by Recomb. Complex Cleavage of one strand of DNA by RAG-1 and RAG-2 –transesterification reaction catalyzed by RAG-1/RAG-2 –hairpin structure at end of the coding sequence –flush, 5’-phosphorylated DS break at signal sequence Cutting of the hairpin to generate P-nucleotides Trimming of a few nucleotides by a ss-DNA endonuclease Optional addition of N-nucleotides (up to 15) by Tdt Ligation by normal DS-break repair enzymes

6. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

7. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

8. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E VDJ joining and NHEJ –Essentially Rag-generated DS-breaks DS-break repair –end processing (+TdT) –ligation –New patient with SCID (T-B-) –But, all “known” repair enzymes present –All exogenously supplied enzymes failed to complement

9. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Cells 2BN (patient skin primary fibroblast) 2BNneo (SV-40 transformed but not immortal) 2BNhTERT (immortalized by telomerase) 1BR, 1BRneo, 1604hTERT (normal individual) 180BR (no ligase IV), AT5Bi (Ataxia- Talangiectasia)

10. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Cells Rearranging capacity –Transfect with RAG-1, RAG-2, substrate –Rearrange: CAM-r/Amp-r, not: Amp-r –Measured “perfect signal joints” by hyb to oligo probe –Signal joints normally perfect, coding joints: P and N

11. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

12. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Cells PFGE to measure DSBR –Survival exp + C-14TdR label –Tested fragment entry into gel = no repair

13. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

14. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Tests for enzymes –Immunoprecipitation (anti-Xrcc4): Ligase/adenylate – Ku complex formation gel retardation assay –Western blots (hMre11, hRad50, p95) –DNA-PKcs (phosphorylation of p53 peptide)

15. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

16. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

17. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

18. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Ku-like genes in Prokaryotes Both Eubac’s and Archaebac’s Single gene (not “Ku70 and Ku80) Homodimer? Yes. Consistent Neigborhood association: operons –Ku –Ligase –Primase –Nuclease

19. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

20. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Weller et al. (2000) Science 297:1686 Ku formed homodimer, ring structure Binds to DNA, requires ends Amount proportional to DNA length (like MCM’s) Ku is ykoV, ligase is ykoU Mutants had specific sensitivity to ionizing radiation Minimum DNA binding unit, only barrel of Ku Might interact directly with ligase (or primase, nuclease) No homologs for XRCC4 or DNA-PCcs found

21. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E