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PMID: 9440527 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Identification and characterization of Escherichia coli DNA helicase II mutants that exhibit increased unwinding efficiency.

Journal of bacteriology ·Vol. 180 ·No. 2 ·1998-01-00 ·Pages 377-87

Zhang G, Deng E, Baugh L, Kushner SR

Abstract

Using a combination of both ethyl methanesulfonate and site-directed mutagenesis, we have identified a region in DNA helicase II (UvrD) from Escherichia coli that is required for biological function but lies outside of any of the seven conserved motifs (T. C. Hodgman, Nature 333:22-23, 1988) associated with the superfamily of proteins of which it is a member. Located between amino acids 403 and 409, alterations in the amino acid sequence DDAAFER lead to both temperature-sensitive and dominant uvrD mutations. The uvrD300 (A406T) and uvrD301 (A406V) alleles produce UV sensitivity at 44 degrees C but do not affect sensitivity to methyl methanesulfonate (MMS). In contrast, the uvrD303 mutation (D403AD404A) causes increased sensitivity to both UV and MMS and is dominant to uvrD+ when present at six to eight copies per cell. Several of the alleles demonstrated a strong antimutator phenotype. In addition, conjugal recombination is reduced 10-fold in uvrD303 strains. Of all of the amino acid substitutions tested, only an alanine-to-serine change at position 406 (uvrD302) was neutral. To determine the biochemical basis for the observed phenotypes, we overexpressed and purified the UvrD303 protein from a uvrD delta294 deletion background and characterized its enzymatic activities. The highly unusual UvrD303 protein exhibits a higher specific activity for ATP hydrolysis than the wild-type control, while its Km for ATP binding remains unchanged. More importantly, the UvrD303 protein unwinds partial duplex DNA up to 10 times more efficiently than wild-type UvrD. The DNA binding affinities of the two proteins appear comparable. Based on these results, we propose that the region located between amino acids 403 and 409 serves to regulate the unwinding activity of DNA helicase II to provide the proper balance between speed and overall effectiveness in the various DNA repair systems in which the protein participates.

MeSH Terms
Adenosine Triphosphatases/genetics,isolation & purification,metabolism DNA Helicases DNA Topoisomerases, Type I/metabolism Escherichia coli/enzymology,genetics Escherichia coli Proteins Methyl Methanesulfonate/pharmacology Mutation Recombination, Genetic Temperature
Chemicals
Escherichia coli Proteins Methyl Methanesulfonate Adenosine Triphosphatases UvrD protein, E coli DNA Helicases DNA Topoisomerases, Type I
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Zhang G
Department of Genetics, University of Georgia, Athens 30602, USA.
Deng E
Baugh L
Kushner S R
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/JB.180.2.377-387.1998
Published
1998-01-00
Pages
377-87
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC106893
Subset
IM
Grants
NIGMS NIH HHS · GM27997 · United States
NIGMS NIH HHS · GM28760 · United States
External Links
PubMed source DOI: 10.1128/JB.180.2.377-387.1998 PMC Full Text View journal details
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