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