Long-patch base excision DNA repair of 2-deoxyribonolactone prevents the formation of DNA-protein cross-links with DNA polymerase beta.
Oxidized abasic sites are a major form of DNA damage induced by free radical attack and deoxyribose oxidation. 2-Deoxyribonolactone (dL) is a C1'-oxidized abasic site implicated in DNA strand breakage, mutagenesis, and formation of covalent DNA-protein cross-links (DPCs) with repair enzymes such as DNA polymerase beta (polbeta). We show here that mammalian cell-free extracts incubated with Ape1-incised dL substrates under non-repair conditions give rise to DPCs, with a major species dependent on the presence of polbeta. DPC formation was much less under repair than non-repair conditions, with extracts of either polbeta-proficient or -deficient cells. Partial base excision DNA repair (BER) reconstituted with purified enzymes demonstrated that Flap endonuclease 1 (FEN1) efficiently excises a displaced oligonucleotide containing a 5'-terminal dL residue, as would be produced during long-patch (multinucleotide) BER. Simultaneous monitoring of dL repair and dL-mediated DPC formation demonstrated that removal of the dL residue through the combined action of strand-displacement DNA synthesis by polbeta and excision by FEN1 markedly diminished DPC formation with the polymerase. Analysis of the patch size distribution associated with DNA repair synthesis in cell-free extracts showed that the processing of dL residues is associated with the synthesis of >or=2 nucleotides, compared with predominantly single nucleotide replacement for regular abasic sites. Our observations reveal a cellular repair process for dL lesions that avoids formation of DPCs that would threaten the integrity of DNA and perhaps cell viability.
Sung
Jung-Suk
JS
Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
DeMott
Michael S
MS
Demple
Bruce
B
D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D045585:Flap Endonucleases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D011506:Proteins / Q000378:metabolism*; D013400:Sugar Acids / Q000378:metabolism*
D003432:Cross-Linking Reagents; D011506:Proteins; D013400:Sugar Acids; C056405:2,4,5-trihydroxypentanoic acid gamma-lactone; D004247:DNA; D019951:DNA Polymerase beta; D045585:Flap Endonucleases; C468317:FEN1 protein, human