Abstract
The first step of homology-dependent repair of DNA double-strand breaks is the strand-specific processing of DNA ends to generate 3' single-strand tails. Despite its importance, the molecular mechanism underlying end processing is poorly understood in eukaryotic cells. We have taken a biochemical approach to investigate DNA end processing in nucleoplasmic extracts derived from the unfertilized eggs of Xenopus laevis. We found that double-strand DNA ends are specifically degraded in the 5' --> 3' direction in this system. The reaction consists of two steps: an ATP-dependent unwinding of double-strand ends and an ATP-independent 5' --> 3' degradation of single-strand tails. We also found that the Xenopus Werner syndrome protein, a member of the RecQ helicase family, plays an important role in DNA end processing. Mechanistically, Xenopus Werner syndrome protein (xWRN) is required for the unwinding of DNA ends but not for the degradation of single-strand tails. The xWRN-mediated end processing is remarkably similar to the end processing that has been proposed for the Escherichia coli RecQ helicase and RecJ single-strand nuclease, suggesting that this mechanism might be conserved in prokaryotes and eukaryotes.
Highlights
The first step of homology-based repair is the processing of DSBs into 3Ј single-stranded tails
The E. coli RecQ helicase and RecJ ss-DNA exonuclease have been proposed to act in a similar way to process DNA ends, suggesting that a conserved DNA end processing pathway might be present in prokaryotes and eukaryotes
The 32P label was largely retained on the beads, but upon heat treatment, the released labeled DNA was mostly of small size (4 –11 nucleotides). (The small amount of dAMP generated was later converted to dATP, presumably by adenylate kinase and nucleoside diphosphate kinase in nucleoplasmic extract (NPE).) Taken together, these results strongly suggest that ds-DNA end processing in NPE is strand specific and proceeds mostly in the 5Ј 3 3Ј direction
Summary
To better understand homology-dependent DSB repair and the role of xWRN in it, we have initiated a biochemical analysis of DNA end processing using NPE as the model system. We have found that NPE contains robust activity for 5Ј strand-specific processing of double-strand DNA ends This reaction is dependent on ATP and can be separated into two steps: the unwinding of DNA ends and the degradation of the 5Ј ss-tail. We have found that the xWRN helicase plays an important role in DNA end processing by promoting end unwinding. These results provide the first biochemical evidence that DNA end processing in eukaryotes is a coupled end unwinding/5Ј ss-tail degradation reaction and that WRN, a member of RecQ helicase family, acts as the primary helicase for unwinding of ds-DNA ends in NPE. The E. coli RecQ helicase and RecJ ss-DNA exonuclease have been proposed to act in a similar way to process DNA ends, suggesting that a conserved DNA end processing pathway might be present in prokaryotes and eukaryotes
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