Conserved Domain Architecture of Human BLM Helicase Maintains Balance between D-Loop Disruption and Extension

Abstract

Cells must protect their genome from harmful alterations, caused directly by DNA damage or indirectly by inappropriate repair, to avoid cell death or cancerous transformation. DNA double-strand (ds) breaks, the most severe type of DNA damage, are repaired in a potentially error-free manner by homologous recombination (HR). During HR, broken dsDNA ends are transformed into 3' single-stranded (ss) overhangs that form nucleoprotein filaments with recombinases. These filaments can search for intact, homologous dsDNA regions that serve as template for repair and can invade into the homologous DNA molecule to form a displacement loop (D-loop). D-loops are thus key HR DNAintermediate structures. D-loops are thought to be processed by multiple pathways with different outcomes and can serve as early decision points in HR regulation. Members of the RecQ helicase family are implicated both in D-loop stabilization and disruption. The human Bloom's syndrome RecQ helicase (BLM) is thought to channel HR into error-free pathways partially by processing D-loops. However, the exact mechanism of this action is poorly understood. Via a novel kinetic assay, here we show that the conserved domain architecture of BLM maintains an almost 1:1 balance in D-loop disruption and stabilization. Interestingly, the activity profile of BLM markedly differs from that of the very effective D-loop disruptor Escherichia coli RecQ. However, our single-molecule magnetic tweezers experiments reveal that the similar domain architecture of BLM and RecQ, while having different roles in D-loop processing, allows both proteins to sense DNA substrate geometry and unwind dsDNA in a repetitive fashion. Our results highlight how functions of the conserved architecture of RecQ helicases evolved to specialize these enzymes to different cellular requirements.