Mutant forms of the Escherichia colibeta sliding clamp that distinguish between its roles in replication and DNA polymerase V-dependent translesion DNA synthesis.
The Escherichia colibeta sliding clamp is proposed to play an important role in regulating DNA polymerase traffic at the replication fork. As part of an ongoing effort to understand how organisms manage the actions of their multiple DNA polymerases, we examined the ability of several mutant forms of the beta clamp to function in DNA polymerase V- (pol V-) dependent translesion DNA synthesis (TLS) in vivo. Our results indicate that a dnaN159 strain, which expresses a temperature sensitive form of the beta clamp, was impaired for pol V-dependent TLS at the permissive temperature of 37 degrees C. This defect was complemented by a plasmid that expressed near-physiological levels of the wild-type clamp. Using a dnaN159 mutant strain, together with various plasmids expressing mutant forms of the clamp, we determined that residues H148 through R152, which comprise a portion of a solvent exposed loop, as well as position P363, which is located in the C-terminal tail of the beta clamp, are critically important for pol V-dependent TLS in vivo. In contrast, these same residues appear to be less critical for pol III-dependent replication. Taken together, these findings indicate that: (i) the beta clamp plays an essential role in pol V-dependent TLS in vivo and (ii) pol III and pol V interact with non-identical surfaces of the beta clamp.
Sutton
Mark D
MD
Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, SUNY, 3435 Main Street, 140 Farber Hall, Buffalo, NY 14214, USA. mdsutton@Buffalo.edu
Duzen
Jill M
JM
Maul
Robert W
RW
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis; D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D013696:Temperature
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III; C120926:DNA polymerase V, E coli; D004259:DNA-Directed DNA Polymerase