DNA polymerase κ (Polκ), one of the typical member of the Y-family DNA polymerases, has been demonstrated to bypass the 10S(+)-trans-anti-benzo[a]pyrene diol epoxide-N(2)-deoxyguanine adducts (BPDE-dG) efficiently and accurately. A large structural gap between the core and little finger as well as an N-clasp domain are essential to its unique translesion capability. However, whether the extreme N-terminus of Polκ is required for its activity is unclear. In this work, we constructed two mouse Polκ deletions, which have either a catalytic core (mPolκ1-516) or a core without the first 21-residues (mPolκ22-516), and tested their activities in the replication of normal and BPDE-DNA. These two Polκ deletions are nearly as efficient as the full length protein (Polκ1-852) in normal DNA synthesis. However, steady-state kinetics reveals a significant reduction in efficiency of dCTP incorporation opposite the lesion by Polκ22-516, along with increased frequencies for misinsertion compared with Polκ1-852 The next nucleotide insertion opposite the template C immediately following the BPDE-dG was also examined, and the bypass differences induced by deletions were highlighted in both insertion and extension step. We conclude that the extreme N-terminal part of Polκ is required for the processivity and fidelity of Polκ during translesion synthesis of BPDE-dG lesions.
Keywords: BPDE-dG lesions; Y-family DNA polymerase κ; enzyme fidelity; polycyclic aromatic hydrocarbons; translesion DNA synthesis.
© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.