Sliding Clamp–DNA Interactions Are Required for Viability and Contribute to DNA Polymerase Management in Escherichia coli

Abstract

SUMMARY Sliding clamp proteins topologically encircle DNA and play vital roles in coordinating the actions of various DNA replication, repair, and damage tolerance proteins. At least three distinct surfaces of the E. coli β clamp interact physically with the DNA that it topologically encircles. We utilized mutant β clamp proteins bearing G66E and G174A substitutions (β159), affecting the single strand (ss) DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 (β148–152), affecting the double strand (ds) DNA binding region, in order to determine the biological relevance of clamp-DNA interactions. As part of this work, we solved the x-ray crystal structure of β148–152, which verified that the poly-Ala substitutions failed to significantly alter the tertiary structure of the clamp. Based on functional assays, both β159 and β148–152 were impaired for loading and retention on a linear primed DNA in vitro. In the case of β148–152, this defect was not due to altered interactions with the DnaX clamp loader, but rather was the result of impaired β148–152-DNA interactions. Once loaded, β148–152 was proficient for Pol III replication in vitro. In contrast, β148–152 was severely impaired for Pol II and Pol IV replication, and was similarly impaired for direct physical interactions with these Pols. Despite its ability to support Pol III replication in vitro, β148–152 was unable to support viability of E. coli. Nevertheless, physiological levels of β148–152 expressed from a plasmid efficiently complemented the temperature sensitive growth phenotype of a strain expressing β159 (dnaN159), provided that Pol II and Pol IV were inactivated. Although this strain was impaired for Pol V-dependent mutagenesis, inactivation of Pol II and Pol IV restored the Pol V mutator phenotype. Taken together, these results support a model in which a sophisticated combination of competitive clamp-DNA, clamp-partner, and partner-DNA interactions serve to manage the actions of the different E. coli Pols in vivo.

Mutations in the Bacillus subtilis β Clamp That Separate Its Roles in DNA Replication from Mismatch Repair

“…This position is more consistent with a role in protein-protein interaction than with a role in protein stability. It should be noted that the model of the B. subtilis ␤ clamp is elliptical and is not circular like the crystal structures of the E. coli ␤ clamp (24,31,39). This is because our B. subtilis ␤ clamp model is based upon the elliptical Gram-positive S. pneumoniae ␤ clamp structure (www .rcdb.org; PDB accession no.…”

“…The ␤ clamp has an N-terminal face and a C-terminal face, and most current models predict that proteins that bind the ␤ clamp do so on the C-terminal face. The C-terminal face contains the hydrophobic cleft, which serves as a critical binding site for many proteins (5,8,14,18,30,31,48,49,62,73). All three missense mutations isolated as intragenic suppressors of the temperature sensitivity caused by dnaN5(G73R) are predicted to change amino acid residues located on the surface of the ␤ clamp, and two suppressors are located near the hydrophobic cleft (Fig.…”

“…The roles of the E. coli ␤ clamp in translesion synthesis are well established (5,8,30,31). Binding sites on the E. coli ␤ clamp that accommodate translesion polymerases pol IV (DinB) and pol V (UmuD 2 ЈC) have been identified, and the consequence of disrupting their association with the ␤ clamp has illustrated the critical importance of the ␤ clamp to the activity of both of these polymerases (4,5,8,26,30,31,48,49).In addition to the involvement of the ␤ clamp in replication initiation, DNA replication, and translesion synthesis, the E. coli and B. subtilis ␤ clamp also functions in DNA mismatch repair (MMR) (45,46,64). The MMR pathway recognizes and repairs DNA polymerase errors, contributing to the overall fidelity of the DNA replication pathway (reviewed in references 42 and 60).…”

“…The roles of the E. coli ␤ clamp in translesion synthesis are well established (5,8,30,31). Binding sites on the E. coli ␤ clamp that accommodate translesion polymerases pol IV (DinB) and pol V (UmuD 2 ЈC) have been identified, and the consequence of disrupting their association with the ␤ clamp has illustrated the critical importance of the ␤ clamp to the activity of both of these polymerases (4,5,8,26,30,31,48,49).…”

“…In E. coli, many dnaN alleles have been examined and used to define the mechanistic roles of the ␤ clamp in vivo (5,18,24,30,31,48,49,73). A limitation in studying the mechanistic roles of the B. subtilis ␤ clamp is that only two dnaN alleles (␤ clamp) are available, dnaN5 and dnaN34 (36) (www.bgsc.org/), and both of these alleles do not support growth at temperatures above 49°C, suggesting that they may cause similar defects (36) (www .bgsc.org/).…”