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Efficient and High Fidelity Incorporation of dCTP Opposite 7,8‐Dihydro‐8‐oxodeoxyguanosine by Sulfolobus solfataricus DNA Polymerase Dpo4
Author(s) -
Zang Hong,
Irimia Adriana,
Choi JeongYun,
Angel Karen,
Loukachevitch Lioudmila,
Egli Martin,
Guengerich F. Peter
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a39-c
Subject(s) - sulfolobus solfataricus , oligonucleotide , dna polymerase , polymerase , microbiology and biotechnology , dna , chemistry , stereochemistry , biology , biochemistry , gene , archaea
DNA polymerases insert dATP opposite the oxidative damage product 7,8 dihydro‐8‐oxodeoxyguanosine (8‐oxoG) instead of dCTP, to the extent of > 90% with some polymerases. Steady‐state kinetics with the Y‐family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 25‐fold higher incorporation efficiency of dCTP > dATP opposite 8‐oxoG and 4‐fold higher efficiency of extension beyond an 8‐oxoG:C pair than an 8‐oxoG:A pair. The catalytic efficiency for these events with dCTP was similar for G and 8‐oxoG templates. Mass spectral analysis of extended DNA primers showed ≥ 95% incorporation of dCTP > dATP opposite 8‐oxoG. Pre‐steady‐state kinetics showed faster rates of dCTP incorporation opposite 8‐oxoG than G. The measured K d,dCTP was 15‐fold lower for an oligonucleotide containing 8‐oxoG than with G. Extension beyond an 8‐oxoG:C pair was similar to G:C and faster than for an 8‐oxoG:A pair, in contrast to other polymerases. The E a for dCTP insertion opposite 8‐oxoG was lower than opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8‐oxG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca 2+ . The 8‐oxoG:C pair showed classic Watson‐Crick geometry; the 8‐oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with the 8‐oxoG. With dGTP placed opposite 8‐oxoG, pairing was not to the 8‐oxoG but to the 5′ C (and in classic Watson‐Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays.