Mechanism of D-Loop Disruption by the Human Bloom's Syndrome Helicase

Abstract

The most toxic form of DNA damage is the double-stranded DNA break (DSB). To avoid the harmful consequences of DSBs, cells use homologous recombination (HR)-based error-free DNA repair mechanisms. HR processes must be highly organized and regulated in order to take place in the right context, because illegitimate HR and incorrect DNA repair may cause severe genetic abnormalities that can lead to cell death or different types of cancer. Human Bloom's syndrome DNA helicase (BLM), a member of RecQ family, plays crucial roles in HR progression and regulation. In the early steps of HR a three-stranded complex DNA structure, a displacement loop (D-loop), is generated by the strand exchange activity of Rad51 recombinase. The formation of D-loops is essential for HR progression. BLM is able to perform quality control of HR by disrupting D-loops. To investigate the mechanism of D-loop disruption we generated a series of truncated mutants of BLM and set up a gel based fluorimetric assay to monitor the dissolution kinetics under single round conditions. Interestingly we found that all of the investigated constructs are able to disrupt D-loops. Moreover, our quantitative analysis revealed that the different constructs use distinct processing mechanisms. Our results demonstrate how BLM is capable of regulating HR by the dissolution of D-loops and how the different domains present in BLM regulate D-loop processing.