Characterization of novel alleles of the Escherichia coli umuDC genes identifies additional interaction sites of UmuC with the beta clamp.
Translesion synthesis is a DNA damage tolerance mechanism by which damaged DNA in a cell can be replicated by specialized DNA polymerases without being repaired. The Escherichia coli umuDC gene products, UmuC and the cleaved form of UmuD, UmuD', comprise a specialized, potentially mutagenic translesion DNA polymerase, polymerase V (UmuD'(2)C). The full-length UmuD protein, together with UmuC, plays a role in a primitive DNA damage checkpoint by decreasing the rate of DNA synthesis. It has been proposed that the checkpoint is manifested as a cold-sensitive phenotype that is observed when the umuDC gene products are overexpressed. Elevated levels of the beta processivity clamp along with elevated levels of the umuDC gene products, UmuD'C, exacerbate the cold-sensitive phenotype. We used this observation as the basis for genetic selection to identify two alleles of umuD' and seven alleles of umuC that do not exacerbate the cold-sensitive phenotype when they are present in cells with elevated levels of the beta clamp. The variants were characterized to determine their abilities to confer the umuD'C-specific phenotype UV-induced mutagenesis. The umuD variants were assayed to determine their proficiencies in UmuD cleavage, and one variant (G129S) rendered UmuD noncleaveable. We found at least two UmuC residues, T243 and L389, that may further define the beta binding region on UmuC. We also identified UmuC S31, which is predicted to bind to the template nucleotide, as a residue that is important for UV-induced mutagenesis.
Beuning
Penny J
PJ
Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA. beuning@neu.edu
Chan
Sarah
S
Waters
Lauren S
LS
Addepalli
Haripriya
H
Ollivierre
Jaylene N
JN
Walker
Graham C
GC
D015153:Blotting, Western; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology; D016296:Mutagenesis / Q000235:genetics / Q000528:radiation effects; D020125:Mutation, Missense; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013696:Temperature; D014466:Ultraviolet Rays
D029968:Escherichia coli Proteins; C078202:UmuC protein, E coli; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli