Abstract
Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3âČOH (3âČDNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3âČDNA, fluorescent ÎČ and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5âČphosphate (5âČP) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5âČDNA). The 5âČP groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3âČDNA. Interestingly, the Ï subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5âČDNA, showing that Ï·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3âČDNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role.
Highlights
Sliding clamps and clamp loaders play a crucial role in a variety of processes in DNA replication and repair
In order to be effective for DNA replication and repair, †must be loaded onto p/t junctions with a recessed 3 -end (3 DNA)
To determine how both the structure of DNA and the presence of single-stranded binding proteins (SSBs) contribute to DNA specificity by ℠complex, a fluorescent †closing assay was used to report on productive clamp loading events [45]
Summary
Sliding clamps and clamp loaders play a crucial role in a variety of processes in DNA replication and repair. Because of their vital nature, sliding clamps and clamp loaders are present in all kingdoms of life [1]. Sliding clamps are ring shaped proteins that are loaded around DNA in an adenosine triphosphate (ATP)-dependent reaction catalyzed by clamp loaders [1,2,3]. In the presence of a sliding clamp, DNA polymerase is tethered to the parental DNA, preventing dissociation and increasing the rate of overall DNA synthesis [4,5]
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