Reconstitution of human base excision repair with purified proteins.
Base excision repair is a major mechanism for correcting aberrant DNA bases. We are using an in vitro base excision repair assay to fractionate and purify proteins from a human cell extract that are involved in this type of repair. Three fractions are required to reconstitute base excision repair synthesis using a uracil-containing DNA as a model substrate. We previously showed that one fraction corresponds to DNA polymerase beta. A second fraction was extensively purified and found to possess uracil-DNA glycosylase activity and was identified as the product of the UNG gene. A neutralizing antibody to the human UNG protein inhibited base excision repair in crude extract by at least 90%. The third fraction was highly purified and exhibited apurinic/apyrimidinic (AP) endonuclease activity. Immunoblot analysis identified HAP1 as the major polypeptide in fractions possessing DNA repair activity. Recombinant versions of UNG, HAP1, and DNA polymerase beta were able to substitute for the proteins purified from human cells. Addition of DNA ligase I led to ligated repair products. Thus, complete base excision repair of uracil-containing DNA was achieved by a combination of UNG, HAP1, DNA polymerase beta, and DNA ligase I. This is the first complete reconstitution of base excision repair using entirely eukaryotic proteins.
Nicholl
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The Picower Institute for Medical Research, Manhasset, New York 11030, USA.
Nealon
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Kenny
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D000906:Antibodies / Q000494:pharmacology; D001483:Base Sequence; D019757:Carbon-Oxygen Lyases; D045647:DNA Glycosylases; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008190:Lyases / Q000378:metabolism*; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D051981:Uracil-DNA Glycosidase
D000906:Antibodies; D004791:Enzyme Inhibitors; C000607892:LIG1 protein, human; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; D004256:DNA Polymerase I; D043223:Deoxyribonuclease IV (Phage T4-Induced); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D008190:Lyases; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D011088:DNA Ligases; D000072481:DNA Ligase ATP