Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein.
Repair of a uracil-guanine base pair in DNA has been reconstituted with the recombinant human proteins uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta and DNA ligase III. The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch. We show that XRCC1 interacts directly with DNA polymerase beta using far Western blotting, affinity precipitation and yeast two-hybrid analyses. In addition, a complex formed between DNA polymerase beta and a double-stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation assay. The region of interaction with DNA polymerase beta is located within residues 84-183 in the N-terminal half of the XRCC1 protein, whereas the C-terminal region of XRCC1 is involved in binding DNA ligase III. These data indicate that XRCC1, which has no known catalytic activity, might serve as a scaffold protein during base excision-repair. DNA strand displacement and excessive gap filling during DNA repair were observed in cell-free extracts of an XRCC1-deficient mutant cell line, in agreement with the results from the reconstituted system.
Kubota
Y
Y
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, UK.
Nash
R A
RA
Klungland
A
A
Schär
P
P
Barnes
D E
DE
Lindahl
T
T
D001482:Base Composition; D001483:Base Sequence; D015153:Blotting, Western; D002474:Cell-Free System; D045647:DNA Glycosylases; D004256:DNA Polymerase I / Q000302:isolation & purification / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D043223:Deoxyribonuclease IV (Phage T4-Induced); D006147:Guanine; D006801:Humans; D008190:Lyases / Q000378:metabolism; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014498:Uracil; D051981:Uracil-DNA Glycosidase; D000076105:X-ray Repair Cross Complementing Protein 1
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D014498:Uracil; D006147:Guanine; D004256:DNA Polymerase I; D043223:Deoxyribonuclease IV (Phage T4-Induced); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase