期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：50
页码：E6852-E6861
DOI：10.1073/pnas.1518028112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceDNA helicases are essential enzymes that unwind dsDNA to ssDNA to access the information encoded within those strands. We describe a new single-molecule imaging procedure to follow DNA unwinding in real-time and apply it to Escherichia coli RecQ helicase. Using a fluorescent sensor to detect ssDNA and fluorescence microscopy, we observe that DNA unwinding occurs by initial nucleation of RecQ at random DNA sites and concomitant local melting of duplex DNA. Subsequently, RecQ assembles into a distribution of multimeric species that processively unwind DNA at rates proportional to their assembled state. Thus, RecQ helicase acts by a mechanism that is distinctive in that the active form is a variable and heterogeneous ensemble of loosely coupled monomeric entities. DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40-60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.
