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Abstract
BackgroundThe ubiquitous family of DnaN sliding processivity clamp proteins plays essential roles in DNA replication, DNA repair, and cell cycle progression, in part by managing the actions of the different proteins involved in these processes. Interactions of the homodimeric Escherichia coli β clamp with its known partners involves multiple surfaces, including a hydrophobic cleft located near the C-terminus of each clamp protomer.ResultsA mutant E. coli β clamp protein lacking a functional hydrophobic cleft (βC) complemented the temperature sensitive growth phenotype of a strain bearing the dnaN159 allele, which encodes a thermolabile mutant clamp protein (β159). Complementation was conferred by a βC/β159 heterodimer, and was observed only in the absence of the dinB gene, which encodes DNA polymerase IV (Pol IV). Furthermore, the complemented strain was proficient for umuDC (Pol V) -dependent ultraviolet light (UV) -induced mutagenesis.ConclusionsOur results suggest that a single cleft in the homodimeric E. coli β sliding clamp protein is sufficient to support both cell viability, as well as Pol III, Pol IV, and Pol V function in vivo. These findings provide further support for a model in which different Pols switch places with each other on DNA using a single cleft in the clamp.
Highlights
The ubiquitous family of DnaN sliding processivity clamp proteins plays essential roles in DNA replication, DNA repair, and cell cycle progression, in part by managing the actions of the different proteins involved in these processes
Since the b clamp functions as a homodimer, it contains two such clefts, suggesting it may simultaneously manage the actions of two different partner proteins on DNA by acting as a molecular ‘toolbelt.’ In this model, each partner is bound to a different cleft in the clamp
A heterodimeric form of the clamp bearing a recombinant bC protomer in complex with a wild-type protomer was recently determined to be indistinguishable from the wild-type clamp with respect to its ability to be loaded onto primed DNA, and stimulate processive DNA synthesis by Pol III in vitro [12,13]
Summary
The ubiquitous family of DnaN sliding processivity clamp proteins plays essential roles in DNA replication, DNA repair, and cell cycle progression, in part by managing the actions of the different proteins involved in these processes. Bacterial sliding clamps, termed b or DnaN, are encoded by the dnaN gene, and function as homodimers Like their eukaryotic counterparts, these clamps are loaded onto DNA in an ATP-dependent manner by a multi-subunit clamp loader complex [7]. Since the b clamp functions as a homodimer, it contains two such clefts, suggesting it may simultaneously manage the actions of two different partner proteins on DNA by acting as a molecular ‘toolbelt.’ In this model, each partner is bound to a different cleft in the clamp (see Figure 1A; [9,10,11]) Consistent with this model, a single cleft in the clamp is sufficient to support assembly of the clamp onto DNA, as well as processive replication by the replicative E. coli Pol (Pol III) using an in vitro system reconstituted with purified components, suggesting the other cleft is available for physical interaction with a second partner protein [12,13]
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