| First Author | Liu Y | Year | 2014 |
| Journal | Proc Natl Acad Sci U S A | Volume | 111 |
| Issue | 5 | Pages | 1789-94 |
| PubMed ID | 24449898 | Mgi Jnum | J:206431 |
| Mgi Id | MGI:5550283 | Doi | 10.1073/pnas.1324168111 |
| Citation | Liu Y, et al. (2014) Variants of mouse DNA polymerase kappa reveal a mechanism of efficient and accurate translesion synthesis past a benzo[a]pyrene dG adduct. Proc Natl Acad Sci U S A 111(5):1789-94 |
| abstractText | DNA polymerase kappa (Polkappa) is the only known Y-family DNA polymerase that bypasses the 10S (+)-trans-anti-benzo[a]pyrene diol epoxide (BPDE)-N(2)-deoxyguanine adducts efficiently and accurately. The unique features of Polkappa, a large structure gap between the catalytic core and little finger domain and a 90-residue addition at the N terminus known as the N-clasp, may give rise to its special translesion capability. We designed and constructed two mouse Polkappa variants, which have reduced gap size on both sides [Polkappa Gap Mutant (PGM) 1] or one side flanking the template base (PGM2). These Polkappa variants are nearly as efficient as WT in normal DNA synthesis, albeit with reduced accuracy. However, PGM1 is strongly blocked by the 10S (+)-trans-anti-BPDE-N(2)-dG lesion. Steady-state kinetic measurements reveal a significant reduction in efficiency of dCTP incorporation opposite the lesion by PGM1 and a moderate reduction by PGM2. Consistently, Polkappa-deficient cells stably complemented with PGM1 GFP-Polkappa remained hypersensitive to BPDE treatment, and complementation with WT or PGM2 GFP-Polkappa restored BPDE resistance. Furthermore, deletion of the first 51 residues of the N-clasp in mouse Polkappa (mPolkappa52-516) leads to reduced polymerization activity, and the mutant PGM252-516 but not PGM152-516 can partially compensate the N-terminal deletion and restore the catalytic activity on normal DNA. However, neither WT nor PGM2 mPolkappa52-516 retains BPDE bypass activity. We conclude that the structural gap physically accommodates the bulky aromatic adduct and the N-clasp is essential for the structural integrity and flexibility of Polkappa during translesion synthesis. |
