Download Document

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