Abstract
Biosynthetic errors and DNA damage introduce mismatches and lesions in DNA that can lead to mutations. These abnormalities are susceptible to correction by a number of DNA repair mechanisms, each of which requires a distinct set of proteins. Escherichia coli DNA helicase II has been demonstrated to function in two DNA repair pathways, methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair. To define further the role of UvrD in DNA repair a site-specific mutant was characterized. The mutation, uvrDQ251E, resides within helicase motif III, a conserved segment of amino acid homology found in a superfamily of prokaryotic and eukaryotic DNA helicases. The UvrD-Q251E protein failed to complement the mutator and ultraviolet light-sensitive phenotypes of a uvrD deletion strain indicating that the mutant protein is inactive in both mismatch repair and excision repair. Biochemical characterization revealed a significant defect in the ability of the mutant enzyme to initiate unwinding at a nick. The elongation phase of the unwinding reaction was nearly normal. Together, the biochemical and genetic data provide evidence that UvrD-Q251E is dysfunctional because the mutant protein fails to initiate unwinding at the nick(s) used to initiate excision and subsequent repair synthesis. These results provide direct evidence to support the notion that helicase II initiates unwinding from a nick in vivo in mismatch repair and excision repair.
Highlights
The processes of replication, recombination, and oligonucleotide excision repair require that duplex DNA be transiently unwound to yield single-stranded DNA1 templates and reaction intermediates
Previous studies have shown that DNA helicase II is required in both methyldirected mismatch repair [7, 9] and UvrABC-mediated nucleo
To define further the functional role of helicase II in nucleotide excision repair and methyl-directed mismatch repair, we have characterized a site-specific uvrD mutant that fails to function in these pathways in vivo
Summary
The processes of replication, recombination, and oligonucleotide excision repair require that duplex DNA be transiently unwound to yield single-stranded DNA (ssDNA) templates and reaction intermediates. Helicase II releases the damaged oligomer and UvrC protein, again presumbably by unwinding the nicked DNA intermediate [13, 14]. There is no direct evidence that helicase II initiates unwinding of the duplex DNA intermediates in mismatch repair or excision repair at the site of the nick in vivo. Sitespecific mutants in motifs I or II (Walker ATPase A and B sites) [29] encode UvrD proteins dramatically compromised in ATPase and helicase activities [30, 31] Expression of these uvrD alleles fails to complement the mutator and UV lightsensitive phenotypes of a uvrD deletion mutant indicating that both methyl-directed mismatch repair and nucleotide excision repair require the ATPase and/or helicase activities of helicase II
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