BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation

Abstract

Bloom syndrome (BS) is a rare genetic disorder characterized by genomic instability and a high predisposition to cancer. The gene defective in BS, BLM, encodes a member of the RecQ family of 3′-5′ DNA helicases, and is proposed to function in recombinational repair during DNA replication. Here, we have utilized single-molecule fluorescence resonance energy transfer microscopy to examine the behaviour of BLM on forked DNA substrates. Strikingly, BLM unwound individual DNA molecules in a repetitive manner, unwinding a short length of duplex DNA followed by rapid reannealing and reinitiation of unwinding in several successions. Our results show that a monomeric BLM can 'measure' how many base pairs it has unwound, and once it has unwound a critical length, it reverses the unwinding reaction through strand switching and translocating on the opposing strand. Repetitive unwinding persisted even in the presence of hRPA, and interaction between wild-type BLM and hRPA was necessary for unwinding reinitiation on hRPA-coated DNA. The reported activities may facilitate BLM processing of stalled replication forks and illegitimately formed recombination intermediates. © 2009 European Molecular Biology Organization | All Rights Reserved.