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J. Bacteriol., Jan 1998, 377-387, Vol 180, No. 2
G Zhang, E Deng, L Baugh and SR Kushner
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.
Copyright © 1998, American Society for Microbiology
Identification and characterization of Escherichia coli DNA helicase II mutants that exhibit increased unwinding efficiency [In Process Citation]
Department of Genetics, University of Georgia, Athens 30602, USA.
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