Structure of the SSB-DNA polymerase III interface and its role in DNA replication |
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Authors: | Marceau Aimee H Bahng Soon Massoni Shawn C George Nicholas P Sandler Steven J Marians Kenneth J Keck James L |
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Institution: | Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706-1532, USA. |
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Abstract: | Interactions between single-stranded DNA-binding proteins (SSBs) and the DNA replication machinery are found in all organisms, but the roles of these contacts remain poorly defined. In Escherichia coli, SSB's association with the χ subunit of the DNA polymerase III holoenzyme has been proposed to confer stability to the replisome and to aid delivery of primers to the lagging-strand DNA polymerase. Here, the SSB-binding site on χ is identified crystallographically and biochemical and cellular studies delineate the consequences of destabilizing the χ/SSB interface. An essential role for the χ/SSB interaction in lagging-strand primer utilization is not supported. However, sequence changes in χ that block complex formation with SSB lead to salt-dependent uncoupling of leading- and lagging-strand DNA synthesis and to a surprising obstruction of the leading-strand DNA polymerase in vitro, pointing to roles for the χ/SSB complex in replisome establishment and maintenance. Destabilization of the χ/SSB complex in vivo produces cells with temperature-dependent cell cycle defects that appear to arise from replisome instability. |
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Keywords: | DNA replication genome maintenance protein interaction X‐ray crystal structure |
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