Mutations in the Escherichia coli UvrB ATPase motif compromise excision repair capacity

Journal Article · Fri Sep 01 04:00:00 EDT 1989 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.86.17.6577· OSTI ID:5406847

Seeley, T W; Grossman, L ^[1]

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1. Johns Hopkins Univ., Baltimore, MD (USA)

The Escherichia coli UvrB protein possesses an amino acid sequence motif common to many ATPases. The role of this motif in UvrB has been investigated by site-directed mutagenesis. Three UvrB mutants, with amino acid replacements at lysine-45, failed to confer UV resistance when tested in the UV-sensitive strain N364 (delta uvrB), while five other mutants constructed near this region of UvrB confer wild-type levels of UV resistance. Because even the conservative substitution of arginine for lysine-45 in UvrB results in failure to confer UV resistance, we believe we have identified an amino acid side chain in UvrB essential to nucleotide excision repair in E. coli. The properties of two purified mutant UvrB proteins, lysine-45 to alanine (K45A) and asparagine-51 to alanine (N51A), were analyzed in vitro. While the K45A mutant is fully defective in incision of UV-irradiated DNA, K45A is capable of interaction with UvrA in forming an ATP-dependent nucleoprotein complex. The K45A mutant, however, fails to activate the characteristic increase in ATPase activity observed with the wild-type UvrB in the presence of UvrA and DNA. From these results we conclude that there is a second nucleotide-dependent step in incision following initial complex formation, which is defective in the K45A mutant. This experimental approach may prove of general applicability in the study of function and mechanism of other ATPase motif proteins.

• OSTI ID: 5406847
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA) Vol. 86:17; ISSN PNASA; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

560130* -- Radiation Effects on Microorganisms
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ACID ANHYDRASES
AMINO ACID SEQUENCE
ATP-ASE
BACTERIA
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
BIOLOGICAL VARIABILITY
CLONING
DNA REPAIR
DOSE-RESPONSE RELATIONSHIPS
ELECTROMAGNETIC RADIATION
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
EXCISION REPAIR
GENETIC VARIABILITY
HYDROLASES
MICROORGANISMS
MOLECULAR STRUCTURE
MUTANTS
MUTATIONS
PHOSPHOHYDROLASES
RADIATION INDUCED MUTANTS
RADIATIONS
RADIOINDUCTION
RECOVERY
REPAIR
ULTRAVIOLET RADIATION