Novel enzymatic function of DNA polymerase nu in translesion DNA synthesis past major groove DNA-peptide and DNA-DNA cross-links

Abstract

DNA polymerase nu (POLN or pol nu) is a newly discovered A family polymerase that generates a high error rate when incorporating nucleotides opposite dG; its translesion DNA synthesis (TLS) capability has only been demonstrated for high fidelity replication bypass of thymine glycol lesions. In the current investigation, we describe a novel TLS substrate specificity of pol nu, demonstrating that it is able to bypass exceptionally large DNA lesions whose linkages are through the DNA major groove. Specifically, pol nu catalyzed efficient and high fidelity TLS past peptides linked to N(6)-dA via a reduced Schiff base linkage with a gamma-hydroxypropano-dA. Additionally, pol nu could bypass DNA interstrand cross-links with linkage between N(6)-dAs in complementary DNA strands. However, the chemically identical DNA--peptide and DNA interstrand cross-links completely blocked pol nu when they were located in the minor groove via a N(2)-dG linkage. Furthermore, we showed that pol nu incorporated a nucleotide opposite the 1,N(6)-etheno-dA (epsilondA) in an error-free manner and (+)-trans-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide-dA [(+)-BPDE-dA] in an error-prone manner, albeit with a greatly reduced capability. Collectively, these data suggest that although pol nu bypass capacity cannot be generalized to all major groove DNA adducts, this polymerase could be involved in TLS when genomic replication is blocked by extremely large major groove DNA lesions. In view of the recent observation that pol nu may have a role in cellular tolerance to DNA cross-linking agents, our findings provide biochemical evidence for the potential functioning of this polymerase in the bypass of some DNA-protein and DNA-DNA cross-links.

Figure 1

Structures of DNA adducts. The PCLs consist of either a tetra (Lys-Trp-Lys-Lys) or dodecyl (Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr) peptide attached via an acrolein moiety at the N⁶ position of dA (a) or N² position of dG (c). The resulting PCLs are referred to as N⁶-dA PCL4, N⁶-dA PCL12, N²-dG PCL4, and N²-dG PCL12. (b) Structure of N⁶-dA−N⁶-dA interstrand cross-link. (d) Structure of N²-dG−N²-dG interstrand cross-link. (e) Structure of 1,N⁶-etheno-dA. (f) Structure of (+)-trans-anti-BPDE-dA.

Figure 2

Replication bypass of N⁶-dA peptide cross-links by human pol ν. (a) Primer extensions were catalyzed by 50 nM human pol ν under running start conditions (−3 primer) in the presence of 5 nM primer template containing ND, N⁶-dA PCL4, or N⁶-dA PCL12 for 30 min at 37 °C. (b) Single nucleotide incorporations and primer extensions were catalyzed by 5 nM human pol ν under standing start conditions (−1 primer) in the presence of 2 nM primer template containing ND or N⁶-dA PCL12 for 15 min at 37 °C. Reactions shown in lanes 1−6 and lanes 7−12 were conducted side-by-side, and products were separated on the same gel. The reactions shown in panels a and b were carried out in the presence of 100 μM individual or all dNTPs. (c) dTTP titration assays with 2 nM primer template were catalyzed by 5 nM human pol ν under standing start conditions (−1 primer) for 10 min at 37 °C using the same substrates as described in panel b. dTTP concentration ranges from 6.4 nM to 100 μM (5-fold incremental increase). Reactions shown in lanes 1−8 and lanes 9−16 were conducted side-by-side, and products were separated on the same gel.

Figure 3

Replication bypass of N⁶-dA−N⁶-dA interstrand cross-links by human pol ν. Primer extensions were catalyzed by 10 nM human pol ν under running start conditions (−9 primer) using ND, N⁶-dA ICL1 (a), or N⁶-dA ICL2 (b). Reactions shown in lanes 1−2 and lanes 3−4 were conducted side-by-side, and products were separated on the same gel. (c) Single nucleotide incorporations and primer extensions were catalyzed by 5 nM human pol ν under standing start conditions (−1 primer) using ND and N⁶-dA ICL2. All reactions were carried out in the presence of 5 nM primer template and 100 μM individual or all dNTPs for 15 min at 37 °C.

Figure 4

Replication bypass of N²-dG peptide and interstrand cross-links by human pol ν. (a) Primer extensions were catalyzed by 10 nM human pol ν under running start conditions (−4 primer) in the presence of 5 nM primer template containing ND, N²-dG PCL4, or N²-dG PCL12 for 30 min at 37 °C. (b) Primer extensions were catalyzed by 25 nM human pol ν under running start conditions (−10 primer) in the presence of 7.5 nM primer template containing ND, N²-dG ICL1, N²-dG ICL2, N²-dG ICL3, or N²-dG ICL4 for 10 min at 37 °C. All reactions were carried out in the presence of 100 μM dNTPs.

Figure 5

Replication bypass of 1,N⁶-etheno-dA and (+)-trans-anti-BPDE-dA by human pol ν. (a) Primer extensions were catalyzed by 10 nM human pol ν under running start conditions (−3 primer) in the presence of 5 nM primer template containing ND or εdA. (b) Single nucleotide incorporations and primer extensions were catalyzed by 5 nM human pol ν under standing start conditions (−1 primer) in the presence of 5 nM primer template using the same substrates as described in panel a. (c) Primer extensions were catalyzed by 10 nM human pol ν under running start conditions (−6 primer) in the presence of 2 nM primer template containing ND or (+)-BPDE-dA. (d) Single nucleotide incorporations and primer extensions were catalyzed by 5 nM human pol ν under standing start conditions (−1 primer) in the presence of 2 nM primer template using the same substrates as described in panel c. All reactions were carried out in the presence of 100 μM individual or all dNTPs for 15 min at 37 °C.

Figure 6

Replication bypass of N⁶-dA peptide cross-link by yeast pol δ. (a) Single nucleotide incorporations and primer extensions were catalyzed by 0.5 nM yeast pol δ with 2 nM primer template containing ND or N⁶-dA PCL12 under standing start conditions (−1 primer) in the presence or absence of 5 nM PCNA for 20 min at 37 °C. Reactions were carried out in the presence of 100 μM individual or all dNTPs. (b) dTTP titration assays with 2 nM primer template were catalyzed by 0.5 nM yeast pol δ under standing start conditions (−1 primer) in the absence of PCNA for 10 min at 37 °C using the same substrates as described in panel a. dTTP concentration ranges from 6.4 nM to 100 μM (5-fold incremental increase).