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Novel Assay Resolves D-Loop Processing Pathways By E-Coli Recq And Human Blm Helicases

BIOPHYSICAL JOURNAL(2019)

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Abstract
Via the process of homologous recombination (HR) cells can repair various types of DNA damage. Moreover, HR is essential for the generation of genetic diversity in meiosis. In both cases quality control is required to avoid deleterious consequences of imprecise recombination and to regulate the frequency of recombination events. Human Bloom's syndrome (RecQ-family) helicase (BLM) has been implicated in both the quality control and efficient progression of HR events. BLM can disrupt recombination intermediate joint DNA molecules (e.g. D-loops), possibly to selectively inhibit imprecise recombination during repair and to limit the number of chromosome crossovers during meiosis. Intriguingly, BLM is also implicated in the stabilization and extension of D-loops. To resolve the underlying mechanism producing these antagonist behaviors, we developed a new kinetic assay that precisely resolves all possible outcomes of D-loop processing by helicases. By combining this assay with single-molecule DNA unwinding experiments, we show that human BLM maintains a balance between disruption and stabilization of D-loops. Interestingly, the activity profile of BLM markedly differs from that of bacterial (Escherichia coli) RecQ helicase that is strongly biased toward disruption of D-loops. Based on these results we hypothesize that the D-loop disruption or stabilization activities of BLM are selectively utilized by the cell to halt or channel HR into different pathways, respectively. Our assay is also suitable for elucidating the influence of additional factors regulating BLM's predisposition towards different D-loop processing activities.
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Key words
human blm,pathways,assay,d-loop
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