Frameshift deletion by Sulfolobus solfataricus P2 DNA polymerase Dpo4 T239W is selective for purines and involves normal conformational change followed by slow phosphodiester bond formation.
The human DNA polymerase kappa homolog Sulfolobus solfataricus DNA polymerase IV (Dpo4) produces "-1" frameshift deletions while copying unmodified DNA and, more frequently, when bypassing DNA adducts. As judged by steady-state kinetics and mass spectrometry, bypass of purine template bases to produce these deletions occurred rarely but with 10-fold higher frequency than with pyrimidines. The DNA adduct 1,N(2)-etheno-2'-deoxyguanosine, with a larger stacking surface than canonical purines, showed the highest frequency of formation of -1 frameshift deletions. Dpo4 T239W, a mutant we had previously shown to produce fluorescence changes attributed to conformational change following dNTP binding opposite cognate bases (Beckman, J. W., Wang, Q., and Guengerich, F. P. (2008) J. Biol. Chem. 283, 36711-36723), reported similar conformational changes when the incoming dNTP complemented the base following a templating purine base or bulky adduct (i.e. the "+1" base). However, in all mispairing cases, phosphodiester bond formation was inefficient. The frequency of -1 frameshift events and the associated conformational changes were not dependent on the context of the remainder of the sequence. Collectively, our results support a mechanism for -1 frameshift deletions by Dpo4 that involves formation of active complexes via a favorable conformational change that skips the templating base, without causing slippage or flipping out of the base, to incorporate a complementary residue opposite the +1 base, in a mechanism previously termed "dNTP-stabilized incorporation." The driving force is attributed to be the stacking potential between the templating base and the incoming dNTP base.
Zhang
Huidong
H
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Beckman
Jeff W
JW
Guengerich
F Peter
FP
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D016368:Frameshift Mutation; D006801:Humans; D006860:Hydrogen Bonding; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D011687:Purines / Q000378:metabolism*; D048229:Sulfolobus solfataricus / Q000201:enzymology / Q000235:genetics
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D011687:Purines; D019951:DNA Polymerase beta