Analyzing the handoff of DNA from UvrA to UvrB utilizing DNA-protein photoaffinity labeling.
To better define the molecular architecture of nucleotide excision repair intermediates it is necessary to identify the specific domains of UvrA, UvrB, and UvrC that are in close proximity to DNA damage during the repair process. One key step of nucleotide excision repair that is poorly understood is the transfer of damaged DNA from UvrA to UvrB, prior to incision by UvrC. To study this transfer, we have utilized two types of arylazido-modified photoaffinity reagents that probe residues in the Uvr proteins that are closest to either the damaged or non-damaged strands. The damaged strand probes consisted of dNTP analogs linked to a terminal arylazido moiety. These analogs were incorporated into double-stranded DNA using DNA polymerase beta and functioned as both the damage site and the cross-linking reagent. The non-damaged strand probe contained an arylazido moiety coupled to a phosphorothioate-modified backbone of an oligonucleotide opposite the damaged strand, which contained an internal fluorescein adduct. Six site-directed mutants of Bacillus caldotenax UvrB located in different domains within the protein (Y96A, E99A, R123A, R183E, F249A, and D510A), and two domain deletions (Delta2 and Deltabeta-hairpin), were assayed. Data gleaned from these mutants suggest that the handoff of damaged DNA from UvrA to UvrB proceeds in a three-step process: 1) UvrA and UvrB bind to the damaged site, with UvrA in direct contact; 2) a transfer reaction with UvrB contacting mostly the non-damaged DNA strand; 3) lesion engagement by the damage recognition pocket of UvrB with concomitant release of UvrA.
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Matthew J
MJ
DellaVecchia
Deborah L
DL
Croteau
Milan
M
Skorvaga
Sergey V
SV
Dezhurov
Olga I
OI
Lavrik
Bennett
B
Van Houten
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000502:physiology*; D001407:Bacillus / Q000378:metabolism; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry* / Q000528:radiation effects; D004249:DNA Damage; D004265:DNA Helicases / Q000737:chemistry*; D019951:DNA Polymerase beta / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology*; D008027:Light; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013997:Time Factors
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; C058093:UvrB protein, E coli; D004247:DNA; D019951:DNA Polymerase beta; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases