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NHEJ

NHEJ. Cleaning up loose ends. 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

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NHEJ

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  1. NHEJ Cleaning up loose ends... Karp/CELL & MOLECULAR BIOLOGY 3E

  2. Karp/CELL & MOLECULAR BIOLOGY 3E

  3. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

  4. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

  5. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

  6. Karp/CELL & MOLECULAR BIOLOGY 3E

  7. Karp/CELL & MOLECULAR BIOLOGY 3E

  8. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

  9. 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) Karp/CELL & MOLECULAR BIOLOGY 3E

  10. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

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  12. Cells • PFGE to measure DSBR • Survival exp + C-14TdR label • Tested fragment entry into gel = no repair Karp/CELL & MOLECULAR BIOLOGY 3E

  13. Karp/CELL & MOLECULAR BIOLOGY 3E

  14. Tests for enzymes • 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) Karp/CELL & MOLECULAR BIOLOGY 3E

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  17. Karp/CELL & MOLECULAR BIOLOGY 3E

  18. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

  19. Karp/CELL & MOLECULAR BIOLOGY 3E

  20. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

  21. Karp/CELL & MOLECULAR BIOLOGY 3E

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