title: Modulation of UvrD helicase activity by covalent DNA-protein cross-links.
abstract: UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3' to 5' direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD interactions with proteins bound to DNA have significant biological implications, the effects of covalent DNA-protein cross-links on UvrD helicase activity have not been characterized. Herein, we demonstrate that UvrD-catalyzed strand separation was inhibited on a DNA strand to which a 16-kDa protein was covalently bound. Our sequestration studies suggest that the inhibition of UvrD activity is most likely due to a translocation block and not helicase sequestration on the cross-link-containing DNA substrate. In contrast, no inhibition of UvrD-catalyzed strand separation was apparent when the protein was linked to the complementary strand. The latter result is surprising given the earlier observations that the DNA in this covalent complex is severely bent ( approximately 70 degrees ), with both DNA strands making multiple contacts with the cross-linked protein. In addition, UvrD was shown to be required for replication of plasmid DNAs containing covalent DNA-protein complexes. Combined, these data suggest a critical role for UvrD in the processing of DNA-protein cross-links.
authors.affiliation: Department of Molecular and Medical Genetics, Oregon Health & Science University, Center for Research on Occupational and Environmental Toxicology, Portland, Oregon 97239, USA.
authors.forename: Anuradha
authors.initials: A
authors.lastname: Kumari
authors.forename: Irina G
authors.initials: IG
authors.lastname: Minko
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Smith
authors.forename: R Stephen
authors.initials: RS
authors.lastname: Lloyd
authors.forename: Amanda K
authors.initials: AK
authors.lastname: McCullough
mesh_terms: D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004265:DNA Helicases / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D011506:Proteins; D004247:DNA; C506746:UvrD protein, E coli; D004265:DNA Helicases
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