BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation.
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.
Yodh
Jaya G
JG
Department of Physics, Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Stevens
Benjamin C
BC
Kanagaraj
Radhakrishnan
R
Janscak
Pavel
P
Ha
Taekjip
T
D001816:Bloom Syndrome / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics; D004260:DNA Repair; D004261:DNA Replication; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D053484:RecQ Helicases / Q000378:metabolism*
D009841:Oligonucleotides; D004247:DNA; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases