Steric Gate Variants of UmuC Confer UV Hypersensitivity onEscherichia coli

Abstract

Y family DNA polymerases are specialized for replication of damaged DNA and represent a major contribution to cellular resistance to DNA lesions. Although the Y family polymerase active sites have fewer contacts with their DNA substrates than replicative DNA polymerases, Y family polymerases appear to exhibit specificity for certain lesions. Thus, mutation of the steric gate residue of Escherichia coli DinB resulted in the specific loss of lesion bypass activity. We constructed variants of E. coli UmuC with mutations of the steric gate residue Y11 and of residue F10 and determined that strains harboring these variants are hypersensitive to UV light. Moreover, these UmuC variants are dominant negative with respect to sensitivity to UV light. The UV hypersensitivity and the dominant negative phenotype are partially suppressed by additional mutations in the known motifs in UmuC responsible for binding to the ␤ processivity clamp, suggesting that the UmuC steric gate variant exerts its effects via access to the replication fork. Strains expressing the UmuC Y11A variant also exhibit decreased UV mutagenesis. Strikingly, disruption of the dnaQ gene encoding the replicative DNA polymerase proofreading subunit suppressed the dominant negative phenotype of a UmuC steric gate variant. This could be due to a recruitment function of the proofreading subunit or involvement of the proofreading subunit in a futile cycle of base insertion/excision with the UmuC steric gate variant.

Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

“…The mutations in the ␤-binding sites of UmuC are designated ␤ 361 . These mutations disrupt the binding of UmuC to the ␤ clamp, and therefore, the resultant UmuC should not be recruited to the replication fork (6,65). N32A, N33A, and D34A conferred hypersensitivity to UV radiation when combined with mutations in the ␤-binding motifs in the GW8017 strain (Fig.…”

“…Strains harboring alanine variants of the N-terminal loop 1 residues (residues 32 to 34) were exposed to increasing concentrations of nitrofurazone (NFZ; 5-nitro-2-furaldehyde semicarbazone; TCI America) and 4-nitroquinoline-N-oxide (4-NQO; Acros Organics), as described previously (7,65). Stock solutions (10 mg/ml) were freshly prepared in N,N-dimethyl formamide (Fisher Scientific) and protected from light.…”

“…Western blotting was performed as described previously (65). Strains harboring variants of the N-terminal loop 1 residues 31 to 34 (alanine or conservative mutations) were exposed to 10 J/m 2 UV radiation, while all others were exposed to 25 J/m 2 UV radiation.…”

“…UV survival and mutagenesis assays were performed as described previously (7,65). Variants were exposed to 25 J/m 2 254-nm UV radiation unless otherwise noted for mutagenesis assays.…”

Effects of non‐catalytic, distal amino acid residues on activity of E. coli DinB (DNA polymerase IV)

“…There are several important residues that have previously been identified in DinB [Walsh et al, 2011b], including the ''steric gate'' residue (F13), mutation of which eliminates the enhanced ability of DinB to bypass its cognate lesion N 2 -furfuryldG . The steric gate residues found in many DNA polymerases [Astatke et al, 1998;Bonnin et al, 1999;Ogawa et al, 2001;Yang et al, 2002;DeLucia et al, 2003;Shurtleff et al, 2009;Sherrer et al, 2010], including DinB , prevent ribonucleotide incorporation [Patel et al, 2000;Brown et al, 2011]. Residue Y79, which is near the steric gate residue and highly conserved in the DinB family, is thought to modulate the function of the steric gate residue [Jarosz et al, 2009].…”