Journal
EMBO JOURNAL
Volume 19, Issue 13, Pages 3418-3427Publisher
OXFORD UNIV PRESS
DOI: 10.1093/emboj/19.13.3418
Keywords
DNA unwinding; fluorescence stopped-flow; kinetics; motor protein; protein-DNA interactions
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Funding
- NIGMS NIH HHS [GM55310, R01 GM055310] Funding Source: Medline
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We have investigated the mechanism of binding single-stranded DNA (ssDNA) into the central channel of the ring-shaped T7 gp4A' helicase-primase hexamer. Presteady-state kinetic studies show a facilitated five-step mechanism and provide understanding of how a ring-shaped helicase can be loaded on the DNA during the initiation of replication. The effect of a primase recognition sequence on the observed kinetics suggests that binding to the helicase DNA-binding site is facilitated by transient binding to the primase DNA-binding site, which is proposed to be a loading site. The proposed model involves the fast initial binding of the DNA to the primase site on the outside of the helicase ring, a fast conformational change, a ring-opening step, migration of the DNA into the central channel of the helicase ring, and ring closure. Although an intermediate protein-DNA complex is kinetically stable, only the last species in the five-step mechanism is poised to function as a helicase at the unwinding junction.
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