期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 359, 期 1, 页码 55-65出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2006.02.070
关键词
single-stranded DNA binding protein; protein-DNA interactions; laser temperature-jump kinetics; time-resolved FRET; replication
资金
- NIGMS NIH HHS [GM30498, R01 GM030498] Funding Source: Medline
The Escherichia coli single-stranded DNA binding protein (SSB) binds selectively to single-stranded (ss) DNA intermediates during DNA replication, recombination and repair. Each subunit of the homo-tetrameric protein contains a potential ssDNA binding site, thus the protein can bind to ssDNA in multiple binding modes, one of which is the (SSB)(65) mode, in which a 65 nucleotide stretch of ssDNA interacts with and wraps around all four subunits of the tetramer. Previous stopped-flow kinetic studies of (SSB)(65) complex formation using the oligodeoxynucleotide, (dT)(70), were unable to resolve the initial binding step from the rapid wrapping of ssDNA around the tetramer. Here we report a laser temperature-jump study with resolution in the similar to 500 ns to 4 ms time range, which directly detects these ssDNA wrapping/unwrapping steps. Biphasic time courses are observed with a fast phase that is concentration-independent and which occurs on a time-scale of tens of microseconds, reflecting the wrapping/unwrapping of ssDNA around the SSB tetramer. Analysis of the slower binding phase, in combination with equilibrium binding and stopped-flow kinetic studies, also provides evidence for a previously undetected intermediate along the pathway to forming the (SSB)(65) complex. (c) 2006 Elsevier Ltd. All rights reserved.
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