DNA polymerase ν (POLN or pol ν) 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 ν, demonstrating that it is able to bypass exceptionally large DNA lesions whose linkages are through the DNA major groove. Specifically, pol ν catalyzed efficient and high fidelity TLS past peptides linked to N6-dA via a reduced Schiff base linkage with a γ-hydroxypropano-dA. Additionally, pol ν could bypass DNA interstrand cross-links with linkage between N6-dAs in complementary DNA strands. However, the chemically identical DNA−peptide and DNA interstrand cross-links completely blocked pol ν when they were located in the minor groove via a N2-dG linkage. Furthermore, we showed that pol ν incorporated a nucleotide opposite the 1,N6-etheno-dA (εdA) 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 ν 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 ν 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.

DNA–protein cross-links (adducts) are formed in cellular DNA under a variety of conditions, particularly following exposure to an α,β-unsaturated aldehyde, acrolein. DNA–protein cross-links are subject to repair or damage-tolerance processes. These adducts serve as substrates for proteolytic degradation, yielding DNA–peptide lesions that have been shown to be actively repaired by the nucleotide excision repair complex. Alternatively, DNA–peptide cross-links can be subjected to replication bypass. We present new evidence about the capabilities of DNA polymerases to synthesize DNA past such cross-links. DNAs were constructed with site-specific cross-links, in which either a tetrapeptide or dodecylpeptide was covalently attached at the N2 position of guanine via an acrolein adduct, and replication bypass assays carried out with members of the DinB family of polymerases, human polymerase (pol)κ and Escherichia coli (E. coli) pol IV, and various E. coli polymerases that do not belong to the DinB family. Pol κ was able to catalyze both the incorporation and extension steps with an efficiency that was qualitatively indistinguishable from control (undamaged) substrates. Fidelity was comparable on all these substrates, suggesting that pol κ would have a role in the low mutation frequency associated with replication of these adducts in mammalian cells. When the E. coli orthologue of pol κ, damage-inducible DNA polymerase, pol IV, was analyzed on the same substrates, pause sites were detected opposite and three nucleotides beyond the site of the lesion, with incorporation opposite the lesion being accurate. In contrast, neither E. coli replicative polymerase, pol III, nor E. coli damage-inducible polymerases, pol II and pol V, could efficiently incorporate a nucleotide opposite the DNA–peptide cross-links. Consistent with a role for pol IV in tolerance of these lesions, the replication efficiency of DNAs containing DNA–peptide cross-links was greatly reduced in pol IV-deficient cells. Collectively, these data indicate an important role for the DinB family of polymerases in tolerance mechanisms of N2-guanine linked DNA–peptide cross-links.

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Human DNA polymerase kappa (pol κ) is a translesion synthesis (TLS) polymerase that catalyzes TLS past various minor groove lesions including N 2-dG linked acrolein- and polycyclic aromatic hydrocarbon-derived adducts, as well as N 2-dG DNA–DNA interstrand cross-links introduced by the chemotherapeutic agent mitomycin C. It also processes ultraviolet light-induced DNA lesions. Since pol κ TLS activity can reduce the cellular toxicity of chemotherapeutic agents and since gliomas overexpress pol κ, small molecule library screens targeting pol κ were conducted to initiate the first step in the development of new adjunct cancer therapeutics. A high-throughput, fluorescence-based DNA strand displacement assay was utilized to screen ∼16,000 bioactive compounds, and the 60 top hits were validated by primer extension assays using non-damaged DNAs. Candesartan cilexetil, manoalide, and MK-886 were selected as proof-of-principle compounds and further characterized for their specificity toward pol κ by primer extension assays using DNAs containing a site-specific acrolein-derived, ring-opened reduced form of γ-HOPdG. Furthermore, candesartan cilexetil could enhance ultraviolet light-induced cytotoxicity in xeroderma pigmentosum variant cells, suggesting its inhibitory effect against intracellular pol κ. In summary, this investigation represents the first high-throughput screening designed to identify inhibitors of pol κ, with the characterization of biochemical and biologically relevant endpoints as a consequence of pol κ inhibition. These approaches lay the foundation for the future discovery of compounds that can be applied to combination chemotherapy.