DNA Polymerase POLN Participates in Cross-Link Repair and Homologous Recombination

Abstract

All cells rely on DNA polymerases to duplicate their genetic material and to repair or bypass DNA lesions. In humans, 16 polymerases have been identified, and each bears specific functions in genome maintenance. We identified here the recently discovered polymerase POLN to be involved in repair of DNA cross-links. Such DNA lesions are highly toxic and are believed to be repaired by the sequential activity of nucleotide excision repair, translesion synthesis, and homologous recombination mechanisms. By functionally assaying its role in these processes, we unraveled an unexpected involvement of POLN in homologous recombination. Moreover, we obtained evidence for physical and functional interaction of POLN with factors belonging to the Fanconi anemia pathway, a master regulator of cross-link repair. Finally, we show that POLN interacts and cooperates in DNA repair with the helicase HEL308, which shares a common origin with POLN in the Drosophila mus308 gene. Our data indicate that this novel polymerase-helicase complex participates in homologous recombination repair and is essential for cellular protection against DNA cross-links.Cells are continuously threatened by DNA damage that can potentially alter their genetic information. Numerous overlapping mechanisms exist to deal with DNA lesions. During S phase, unrepaired DNA lesions can block the progression of the replication machinery, causing cell cycle arrest and cell death. Cells have evolved mechanisms that signal the stalling of replication forks and recruit enzymes that can quickly restart these structures. Thus, replication forks can bypass the lesion without removing it. Homologous recombination (HR) and translesion synthesis (TLS) represent the most common mechanisms for dealing with stalled replication forks. In HR, the information on the newly replicated sister chromatid is used to overcome the lesion (32). In contrast, TLS uses special DNA polymerases that are able to replicate damaged DNA. Often, however, TLS polymerases insert wrong nucleotides across lesions, leading to mutations. Most polymerases involved in TLS belong to the Y family of DNA polymerases (2,7,29), which in addition to their characteristic catalytic domains and PCNA interaction protein (PIP) motifs, also have ubiquitinbinding domains toward their C termini. These domains are required for interaction with ubiquitinated PCNA and are essential for their recruitment to stalled replication forks. PCNA is an essential cofactor of DNA polymerases during replication, and it becomes monoubiquitinated when the replication fork is stalled at a damaged site (22). POLN (DNA polymerase ) is a recently discovered enzyme, belonging to the A family of DNA polymerases (16). In vitro, POLN is capable of DNA-templated synthesis, but it shows high mutagenicity, with a preference for inserting T across G and G across T (37). However, its lesion bypass activity is restricted to a subclass of DNA damage, namely, thymine glycol lesions (37). This suggests that POLN might not act as a TLS polymerase in vivo. S...

The ADP-ribosyltransferase PARP10/ARTD10 Interacts with Proliferating Cell Nuclear Antigen (PCNA) and Is Required for DNA Damage Tolerance

“…Functional Assays-Clonogenic assays were perfomed as previously described (39). Two rounds of siRNA were performed, 24 h apart.…”

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

“…Molecular cloning of p125 showed that the catalytic core was highly conserved in evolution from T4 bacteriophage to human Hao et al, 1992;Yang et al, 1992], and Pol d is now classified as a member of the B family polymerases. Closely allied with the early history of Pol d are studies of the eukaryotic DNA-sliding clamp, proliferating cell nuclear antigen (PCNA; [Kuriyan and O'Donnell, 1993;Moldovan et al, 2010]), which was discovered as a processivity factor that stimulated Pol d activity [Prelich et al, 1987].…”

Mouse models of DNA polymerases

“…DSBs can be repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). The DNA polymerases Pol e, Pol b, Pol f, Pol h, Pol y, Pol k, Pol l function in DSB repair by HR or NHEJ [Holbeck et al, 1993;Jessberger et al, 1993;Wang et al, 2004;Xu et al, 2005;Zan et al, 2005;Rattray and Strathern, 2005;Masuda et al, 2006;Nick McElhinny, 2008;Kidane et al, 2010;Moldovan et al, 2010]. The pathways of homologous recombination and nonhomologous end-joining have been reviewed in Chaudhuri and Alt [2004] and San Filippo et al [2008].…”