Evidence for the Involvement of Human DNA Polymerase N in the Repair of DNA Interstrand Cross-Links

Abstract

Human DNA polymerase N (PolN) is an A-family nuclear DNA polymerase whose function is unknown. This study examines the possible role of PolN in DNA repair in human cells treated with PolN-targeted siRNA. HeLa cells with siRNA-mediated knockdown of PolN were more sensitive than control cells to DNA cross-linking agent mitomycin C (MMC), but were not hyper-sensitive to UV irradiation. The MMC hyper-sensitivity of PolN knockdown cells was rescued by the overexpression of DNA polymerase-proficient PolN but not by DNA polymerase-deficient PolN. Furthermore, in vitro experiments showed that purified PolN conducts low efficiency non-mutagenic bypass of a psoralen DNA interstrand cross-link (ICL), whose structure resembles an intermediate in the proposed pathway of ICL repair. These results suggest that PolN might play a role in translesion DNA synthesis during ICL repair in human cells. KeywordsDNA interstrand cross-links; DNA polymerase N; TLS; Psoralen; Chemotherapeutics E. coli PolI is a high fidelity DNA repair polymerase that conducts gap-filling DNA synthesis during nucleotide excision repair (NER), base excision repair (BER), and DNA interstrand cross-link (ICL) repair. E. coli PolI is the prototypical member of A-family DNA polymerases (1). Drosophila melanogaster Mus308 is an A-family nuclear DNA polymerase, the mutants of which are hyper-sensitive to DNA cross-linking agents but not to other DNA damaging agents (2)(3)(4). This suggests that Mus308 may play a role in ICL repair in Drosophila. Two nuclear A-family DNA polymerases, DNA polymerase N (PolN) and DNA polymerase Q (PolQ) were recently discovered (5,6). It has been proposed that PolN and PolQ are mammalian orthologs of Mus308 and that they participate in ICL repair in mammalian cells. However, additional studies are needed to confirm the precise role(s) of PolN and PolQ in DNA repair in mammalian cells.ICLs are generated endogenously as bi-functional products of lipid peroxidation and by exogenous exposure to DNA damaging agents, some of which are commonly used as cancer chemotherapeutic drugs (7,8). Because ICLs covalently link the two complementary strands of duplex DNA, they prevent progression of the DNA replication fork and block RNA transcription. This property makes ICLs highly toxic to proliferating cells. The molecular mechanism of human ICL repair is poorly understood (7-10). The current model of mammalian ICL repair (7-10) suggests that when a DNA replication fork stalls at an ICL, it is recognized *To whom correspondence should be addressed. tbessho@unmc by an endonuclease that generates a double-strand break (DSB) in the vicinity of the ICL (11)(12)(13)(14). Subsequently, XPF-ERCC1 unhooks the ICL, resulting in a gap across from the unhooked ICL (14-17), which cannot be sealed by replicative DNA polymerases δ or ε. Gaps generated during ICL repair are thought to be repaired by translesion DNA synthesis (TLS) or by homologous recombination using the homologous chromosome (the sister chromatid is not available). Once the gap is sea...

Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway

“…POLQ-deficient cells exhibit spontaneous and MMC-induced chromosomal abnormalities, although they are not hypersensitive to cross-linking agents per se (Shima et al 2004). Another A family Pol n (POLN) can perform nonmutagenic bypass of psoralen-induced DNA cross-links, albeit with low efficiency, suggesting a role in TLS during ICL repair (Zietlow et al 2009). However, the biological significance of these in vitro data remains to be established.…”

Advances in Understanding the Complex Mechanisms of DNA Interstrand Cross-Link Repair

“…Polν-knock-down cells are hypersensitive to MMC (Zietlow et al 2009; Moldovan et al 2010). Polν interacts with FANCD2-FANCI as well as RAD51, suggesting a role in replication-dependent ICL repair (Moldovan et al 2010).…”

“…Polν interacts with FANCD2-FANCI as well as RAD51, suggesting a role in replication-dependent ICL repair (Moldovan et al 2010). In vitro, POLν efficiently bypasses major groove ICLs and, with very low efficiency, psoralen ICLs (Zietlow et al 2009; Yamanaka et al 2010). The context in which Polν might operate in ICL repair remains to be determined.…”

Translesion DNA Synthesis and Mutagenesis in Eukaryotes

“…Both enzymes are processive and, given their error rates, it seems likely that their access to DNA is controlled. Mus308 was initially identified through its involvement in tolerance of interstrand cross-linking agents (Harris et al 1996), and the role Pol u and Pol n play in DNA interstrand cross-link repair may reflect their ability to perform TLS or participate in certain forms of homologous recombination (Zietlow et al 2009;Kohzaki et al 2010;Moldovan et al 2010;Yamanaka et al 2010). Importantly, Pol n both physically and functionally interacts with the FA pathway (Moldovan et al 2010).…”