Abstract
Besides X-family DNA polymerases (first of all, Pol β) several other human DNA polymerases from Y- and A- families were shown to possess the dRP-lyase activity and could serve as backup polymerases in base excision repair (Pol ι, Rev1, Pol γ and Pol θ). However the exact position of the active sites and the amino acid residues involved in the dRP-lyase activity in Y- and A- family DNA polymerases are not known. Here we carried out functional analysis of fifteen amino acid residues possibly involved in the dRP-lyase activity of human Pol ι. We show that substitutions of residues Q59, K60 and K207 impair the dRP-lyase activity of Pol ι while residues in the HhH motif of the thumb domain are dispensable for this activity. While both K60G and K207A substitutions decrease Schiff-base intermediate formation during dRP group cleavage, the latter substitution also strongly affects the DNA polymerase activity of Pol ι, suggesting that it may impair DNA binding. These data are consistent with an important role of the N-terminal region in the dRP-lyase activity of Pol ι, with possible involvement of residues from the finger domain in the dRP group cleavage.
Highlights
Human DNA polymerase iota (Pol ι) belongs to the Y-family of translesion DNA polymerases and demonstrates very low accuracy of DNA synthesis
Tested mutations were located in the HhH motif of the thumb domain (K237A, Y244A, K245A, K248A, E251A) and in the fingers domain around the DNA polymerase active site (Y39A, Q59A, K60G, Y61A, K72A, K76A, K77A)
First 420 amino acid residues are sufficient for the DNA polymerase activity of Pol ι and referred to as the catalytic core[1, 20]
Summary
Human DNA polymerase iota (Pol ι) belongs to the Y-family of translesion DNA polymerases and demonstrates very low accuracy of DNA synthesis. In addition to the DNA polymerase activity, human Pol ι has an intrinsic 5′-deoxyribose-5-phosphate lyase activity (dRP-lyase activity)[13, 14]. Pol ι binds to chromatin under oxidative stress and interacts with the BER factor XRCC114. In this work we tested the effects of fifteen amino acid substitutions, including all lysine residues located around the DNA-binding cleft of Pol ι, on its dRP-lyase activity. Tested mutations were located in the HhH motif of the thumb domain (K237A, Y244A, K245A, K248A, E251A) and in the fingers domain around the DNA polymerase active site (Y39A, Q59A, K60G, Y61A, K72A, K76A, K77A). We analyzed the effects of these mutations on Pol ι catalysis and for the first time identified amino acid residues critical for the removal of 5′-dRP group
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