title: Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1).
abstract: Two forms of DNA base excision-repair (BER) have been observed: a 'short-patch' BER pathway involving replacement of one nucleotide and a 'long-patch' BER pathway with gap-filling of several nucleotides. The latter mode of repair has been investigated using human cell-free extracts or purified proteins. Correction of a regular abasic site in DNA mainly involves incorporation of a single nucleotide, whereas repair patches of two to six nucleotides in length were found after repair of a reduced or oxidized abasic site. Human AP endonuclease, DNA polymerase beta and a DNA ligase (either III or I) were sufficient for the repair of a regular AP site. In contrast, the structure-specific nuclease DNase IV (FEN1) was essential for repair of a reduced AP site, which occurred through the long-patch BER pathway. DNase IV was required for cleavage of a reaction intermediate generated by template strand displacement during gap-filling. XPG, a related nuclease, could not substitute for DNase IV. The long-patch BER pathway was largely dependent on DNA polymerase beta in cell extracts, but the reaction could be reconstituted with either DNA polymerase beta or delta. Efficient repair of gamma-ray-induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol beta-dependent long-patch pathway by stimulation of DNase IV.
authors.affiliation: Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, UK.
authors.forename: A
authors.initials: A
authors.lastname: Klungland
authors.forename: T
authors.initials: T
authors.lastname: Lindahl
mesh_terms: D002474:Cell-Free System; D004249:DNA Damage; D011088:DNA Ligases / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D004258:DNA Polymerase III; D004260:DNA Repair; D004277:DNA, Single-Stranded; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004720:Endonucleases; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D045585:Flap Endonucleases; D006801:Humans; D008190:Lyases / Q000378:metabolism; D008957:Models, Genetic; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D010957:Plasmids / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5
chemical_list: D011088:DNA Ligases; D004256:DNA Polymerase I; D004258:DNA Polymerase III; D004277:DNA, Single-Stranded; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004720:Endonucleases; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; D008190:Lyases; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5