The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA
| Autor: | Jaclyn N. Hayner, Lauren G. Douma, Linda B. Bloom |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Saccharomyces cerevisiae Proteins
Protein subunit Saccharomyces cerevisiae DNA Single-Stranded dnaN DNA-binding protein chemistry.chemical_compound Proliferating Cell Nuclear Antigen Replication Protein A Escherichia coli Genetics Phosphorylation Replication Protein C Adenosine Triphosphatases DNA clamp biology Nucleic Acid Enzymes Escherichia coli Proteins DNA Templates Genetic biology.organism_classification Proliferating cell nuclear antigen DNA-Binding Proteins Protein Subunits Clamp chemistry Biochemistry biology.protein Biophysics RNA Protein Binding |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gku774 |
| Popis: | 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 3'OH (3'DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3'DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5'phosphate (5'P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5'DNA). The 5'P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3'DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5'DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3'DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role. |
| Databáze: | OpenAIRE |
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