Fanconi anemia (FA) is a rare genetic disease characterized by congenital defects, bone marrow failure, chromosomal instability, and cancer susceptibility. One hallmark of cells from FA patients is hypersensitivity to interstrand cross-linking agents, such as the chemotherapeutics cisplatin and mitomycin C (MMC). We have recently characterized a FANCD2/FANCI-associated nuclease, KIAA1018/FAN1, the depletion of which sensitizes human cells to these agents. However, as the down-regulation of FAN1 in human cells was mediated by siRNA and thus only transient, we were unable to study the long-term effects of FAN1 loss on chromosomal stability. We now describe the generation of chicken DT40 B cells, in which the FAN1 locus was disrupted by gene targeting. FAN1-null cells are highly sensitive to cisplatin and MMC, but not to ionizing or UV radiation, methyl methanesulfonate, or camptothecin. The cells do not display elevated sister chromatid exchange frequencies, either sporadic or MMC-induced. Interestingly, MMC treatment causes chromosomal instability that is quantitatively, but not qualitatively, comparable to that seen in FA cells. This finding, coupled with evidence showing that DT40 cells deficient in both FAN1 and FANCC, or FAN1 and FANCJ, exhibited increased sensitivity to cisplatin compared with cells lacking only FAN1, suggests that, despite its association with FANCD2/FANCI, FAN1 in DT40 cells participates in the processing of damage induced by interstrand cross-linking-generating agents also independently of the classical FA pathway.

The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases.

Polν and Polθ have specialized functions in immunoglobulin gene rearrangements and only contribute to DNA repair when other homologous recombination–related DNA polymerases are absent.

The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass.