期刊
NUCLEIC ACIDS RESEARCH
卷 44, 期 12, 页码 5849-5860出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkw445
关键词
-
资金
- Burroughs Wellcome Fund Career Award in the Biomedical Sciences
- National Science Foundation [Phy-0404286, Phys-1125844]
- National Science Foundation [IGERT]
- National Physical Science Consortium Fellowship
- W.M. Keck Initiative in the RNA Sciences
- Natural Sciences and Engineering Research Council of Canada and Canada Research Chair Program [299384-2011, 950-213807]
- NSF
- National Institute of Standard and Technology
- Amherst College
- Ministry of Science and Technology of Taiwan [NSC 102-2114-M-002-007]
RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2-4 mu M) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (similar to 4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD-DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5' strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据