Reconstitution of the base excision repair pathway for 7,8-dihydro-8-oxoguanine with purified human proteins.
In mammalian cells, repair of the most abundant endogenous premutagenic lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is initiated by the bifunctional DNA glycosylase OGG1. By using purified human proteins, we have reconstituted repair of 8-oxoG lesions in DNA in vitro on a plasmid DNA substrate containing a single 8-oxoG residue. It is shown that efficient and complete repair requires only hOGG1, the AP endonuclease HAP1, DNA polymerase (Pol) beta and DNA ligase I. After glycosylase base removal, repair occurred through the AP lyase step of hOGG1 followed by removal of the 3'-terminal sugar phosphate by the 3'-diesterase activity of HAP1. Addition of PCNA had a slight stimulatory effect on repair. Fen1 or high concentrations of Pol beta were required to induce strand displacement DNA synthesis at incised 8-oxoG in the absence of DNA ligase. Fen1 induced Pol beta strand displacement DNA synthesis at HAP1-cleaved AP sites differently from that at gaps introduced by hOGG1/HAP1 at 8-oxoG sites. In the presence of DNA ligase I, the repair reaction at 8-oxoG was confined to 1 nt replacement, even in the presence of high levels of Pol beta and Fen1. Thus, the assembly of all the core proteins for 8-oxoG repair catalyses one major pathway that involves single nucleotide repair patches.
Pascucci
B
B
Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Rome, Italy.
Maga
G
G
Hübscher
U
U
Bjoras
M
M
Seeberg
E
E
Hickson
I D
ID
Villani
G
G
Giordano
C
C
Cellai
L
L
Dogliotti
E
E
D001483:Base Sequence; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000378:metabolism; D043382:DNA-Formamidopyrimidine Glycosylase; D004706:Endodeoxyribonucleases / Q000378:metabolism; D045585:Flap Endonucleases; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism*; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051818:Replication Protein C
C453560:7,8-dihydro-8-oxoguanine; D004268:DNA-Binding Proteins; C000607892:LIG1 protein, human; D009841:Oligonucleotides; D018809:Proliferating Cell Nuclear Antigen; D006147:Guanine; D019951:DNA Polymerase beta; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; D051818:Replication Protein C; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D011088:DNA Ligases; D000072481:DNA Ligase ATP