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

Journal Article · Thu Jan 01 00:00:00 EST 2009 · Journal of Molecular Biology

DOI:https://doi.org/10.1016/j.jmb.2009.01.050· OSTI ID:1019807

Heltzel, J; Scouten Ponticelli, S; Sanders, L; Duzen, J; Cody, V; Pace, J; Snell, E; Sutton, M

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 Escherichia coli {beta} clamp interact physically with the DNA that it topologically encircles. We utilized mutant {beta} clamp proteins bearing G66E and G174A substitutions ({beta}159), affecting the single-stranded DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 ({beta}148-152), affecting the double-stranded DNA binding region, to determine the biological relevance of clamp-DNA interactions. As part of this work, we solved the X-ray crystal structure of {beta}148-152, which verified that the poly-Ala substitutions failed to significantly alter the tertiary structure of the clamp. Based on functional assays, both {beta}159 and {beta}148-152 were impaired for loading and retention on a linear primed DNA in vitro. In the case of {beta}148-152, this defect was not due to altered interactions with the DnaX clamp loader, but rather was the result of impaired {beta}148-152-DNA interactions. Once loaded, {beta}148-152 was proficient for DNA polymerase III (Pol III) replication in vitro. In contrast, {beta}148-152 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, {beta}148-152 was unable to support viability of E. coli. Nevertheless, physiological levels of {beta}148-152 expressed from a plasmid efficiently complemented the temperature-sensitive growth phenotype of a strain expressing {beta}159 (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.

• Research Organization: Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
• Sponsoring Organization: DOE - OFFICE OF SCIENCE
• DOE Contract Number: DE-AC02-98CH10886
• OSTI ID: 1019807
• Report Number(s): BNL-95653-2011-JA; JMOBAK; TRN: US201115%%443
• Journal Information: Journal of Molecular Biology, Vol. 387, Issue 1; ISSN 0022-2836
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
BEARINGS
CRYSTAL STRUCTURE
DEFECTS
DNA
DNA POLYMERASES
DNA REPLICATION
ESCHERICHIA COLI
FUNCTIONALS
IN VITRO
IN VIVO
INACTIVATION
MUTAGENESIS
MUTANTS
PHENOTYPE
PLASMIDS
PROTEINS
REPAIR
RESIDUES
RETENTION
STRAINS
SYNTHESIS
VIABILITY
national synchrotron light source