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Second pathway for completion of human DNA base excision‐repair: reconstitution with purified proteins and requirement for DNase IV (FEN1)
Author(s) -
Klungland Arne,
Lindahl Tomas
Publication year - 1997
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/16.11.3341
Subject(s) - biology , dna , deoxyribonuclease i , dna repair , dna glycosylase , base excision repair , microbiology and biotechnology , biochemistry , base sequence
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 β 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 β in cell extracts, but the reaction could be reconstituted with either DNA polymerase β or δ. Efficient repair of γ‐ray‐induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol β‐dependent long‐patch pathway by stimulation of DNase IV.