Long patch base excision repair with purified human proteins. DNA ligase I as patch size mediator for DNA polymerases delta and epsilon.
Among the different base excision repair pathways known, the long patch base excision repair of apurinic/apyrimidinic sites is an important mechanism that requires proliferating cell nuclear antigen. We have reconstituted this pathway using purified human proteins. Our data indicated that efficient repair is dependent on six components including AP endonuclease, replication factor C, proliferating cell nuclear antigen, DNA polymerases delta or epsilon, flap endonuclease 1, and DNA ligase I. Fine mapping of the nucleotide replacement events showed that repair patches extended up to a maximum of 10 nucleotides 3' to the lesion. However, almost 70% of the repair synthesis was confined to 2-4-nucleotide patches and DNA ligase I appeared to be responsible for limiting the repair patch length. Moreover, both proliferating cell nuclear antigen and flap endonuclease 1 are required for the production and ligation of long patch repair intermediates suggesting an important role of this complex in both excision and resynthesis steps.
Pascucci
B
B
Laboratory of Comparative Toxicology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
Stucki
M
M
Jónsson
Z O
ZO
Dogliotti
E
E
Hübscher
U
U
D001483:Base Sequence; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004257:DNA Polymerase II / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D017931:DNA Primers; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D045585:Flap Endonucleases; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism
D017931:DNA Primers; C000607892:LIG1 protein, human; D018809:Proliferating Cell Nuclear Antigen; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D043211:Exodeoxyribonuclease V; D043223:Deoxyribonuclease IV (Phage T4-Induced); D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D011088:DNA Ligases; D000072481:DNA Ligase ATP