Interaction of human DNA polymerase alpha and DNA polymerase I from Bacillus stearothermophilus with hypoxanthine and 8-oxoguanine nucleotides.


To better understand how DNA polymerases interact with mutagenic ...
To better understand how DNA polymerases interact with mutagenic bases, we examined how human DNA polymerase alpha (pol alpha), a B family enzyme, and DNA polymerase from Bacillus stearothermophilus (BF), an A family enzyme, generate adenine:hypoxanthine and adenine:8-oxo-7,8-dihydroguanine (8-oxoG) base pairs. Pol alpha strongly discriminated against polymerizing dATP opposite 8-oxoG, and removing N1, N(6), or N7 further inhibited incorporation, whereas removing N3 from dATP dramatically increased incorporation (32-fold). Eliminating N(6) from 3-deaza-dATP now greatly reduced incorporation, suggesting that incorporation of dATP (analogues) opposite 8-oxoguanine proceeds via a Hoogsteen base pair and that pol alpha uses N3 of a purine dNTP to block this incorporation. Pol alpha also polymerized 8-oxo-dGTP across from a templating A, and removing N(6) from the template adenine inhibited incorporation of 8-oxoG. The effects of N1, N(6), and N7 demonstrated a strong interdependence during formation of adenine:hypoxanthine base pairs by pol alpha, and N3 of dATP again helps prevent polymerization opposite a templating hypoxanthine. BF very efficiently polymerized 8-oxo-dGTP opposite adenine, and N1 and N7 of adenine appear to play important roles. BF incorporates dATP opposite 8-oxoG less efficiently, and modifying N1, N(6), or N7 greatly inhibits incorporation. N(6) and, to a lesser extent, N1 help drive hypoxanthine:adenine base-pair formation by BF. The mechanistic implications of these results showing that different polymerases interact very differently with base lesions are discussed.




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