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The EMBO Journal
(1997) 16, 3341–3348, doi:10.1093/emboj/16.11.3341
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| Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1) |
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Arne Klungland and Tomas Lindahl
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Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK
Received 9 January 1997; Revised 5 February 1997.
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| Abstract |
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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  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 in cell extracts, but the reaction could be reconstituted with either DNA polymerase or  . Efficient repair of  -ray-induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol -dependent long-patch pathway by stimulation of DNase IV. |
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| Keywords: DNA repair, DNase IV, FEN1, oxidative DNA damage, PCNA |
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