Parp‐1 and Parp‐2 are activated by DNA breaks and have been implicated in the repair of DNA single‐strand breaks (SSB). Their involvement in double‐strand break (DSB) repair mediated by homologous recombination (HR) or nonhomologous end joining (NHEJ) remains unclear. We addressed this question using chicken DT40 cells, which have the advantage of carrying only a PARP‐1 gene but not a PARP‐2 gene. We found that PARP‐1 −/− DT40 mutants show reduced levels of HR and are sensitive to various DSB‐inducing genotoxic agents. Surprisingly, this phenotype was strictly dependent on the presence of Ku, a DSB‐binding factor that mediates NHEJ. PARP‐1/KU70 double mutants were proficient in the execution of HR and displayed elevated resistance to DSB‐inducing drugs. Moreover, we found deletion of Ligase IV, another NHEJ gene, suppressed the camptothecin of PARP‐1 −/− cells. Our results suggest a new critical function for Parp in minimizing the suppressive effects of Ku and the NHEJ pathway on HR.

In vertebrates Cdk1 is required to initiate mitosis; however, any functionality of this kinase during S phase remains unclear. To investigate this, we generated chicken DT40 mutants, in which an analog-sensitive mutant cdk1 as replaces the endogenous Cdk1, allowing us to specifically inactivate Cdk1 using bulky ATP analogs. In cells that also lack Cdk2, we find that Cdk1 activity is essential for DNA replication initiation and centrosome duplication. The presence of a single Cdk2 allele renders S phase progression independent of Cdk1, which suggests a complete overlap of these kinases in S phase control. Moreover, we find that Cdk1 inhibition did not induce re-licensing of replication origins in G2 phase. Conversely, inhibition during mitosis of Cdk1 causes rapid activation of endoreplication, depending on proteolysis of the licensing inhibitor Geminin. This study demonstrates essential functions of Cdk1 in the control of S phase, and exemplifies a chemical genetics approach to target cyclin-dependent kinases in vertebrate cells.

The Fanconi anemia pathway is involved in the repair of DNA interstrand cross-links, but it is also involved in regulation of translesion synthesis (TLS). The protein FAAP20 is now identified as a subunit of the Fanconi anemia core complex. FAAP20 interacts with TLS DNA polymerase Rev1, stabilizing Rev1's association with PCNA and promoting DNA damage bypass. The 15 known Fanconi anemia proteins cooperate in a pathway that regulates DNA interstrand cross-link repair. Recent studies indicate that the Fanconi anemia pathway also controls Rev1-mediated translesion DNA synthesis (TLS). We identified Fanconi anemia–associated protein (FAAP20), an integral subunit of the multisubunit Fanconi anemia core complex. FAAP20 binds to FANCA subunit and is required for stability of the complex and monoubiquitination of FANCD2. FAAP20 contains a ubiquitin-binding zinc finger 4 domain and binds to the monoubiquitinated form of Rev1. FAAP20 binding stabilizes Rev1 nuclear foci and promotes interaction of the Fanconi anemia core with PCNA–Rev1 DNA damage bypass complexes. FAAP20 therefore provides a critical link between the Fanconi anemia pathway and TLS polymerase activity. We propose that the Fanconi anemia core complex regulates cross-link repair by channeling lesions to damage bypass pathways and preventing large DNA insertions and deletions.

Protein ubiquitination plays a key role in the regulation of a variety of DNA repair mechanisms. Protein ubiquitination is controlled by the coordinate activity of ubiquitin ligases and deubiquitinating enzymes (DUBs). The deubiquitinating enzyme USP1 regulates DNA repair and the Fanconi anemia pathway through its association with its WD40 binding partner, UAF1, and through its deubiquitination of two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. To investigate the function of USP1 and UAF1, we generated USP1^−/−, UAF1^−/−/−, and USP1^−/−UAF1^−/−/− chicken DT40 cell clones. These three clones showed similar sensitivities to chemical cross-linking agents, to a topoisomerase poison, camptothecin, and to an inhibitor of poly(ADP-ribose) polymerase (PARP), indicating that the USP1/UAF1 complex is a regulator of the cellular response to DNA damage. The hypersensitivity to both camptothecin and a PARP inhibitor suggests that the USP1/UAF1 complex promotes homologous recombination (HR)-mediated double-strand break (DSB) repair. To gain insight into the mechanism of the USP1/UAF1 complex in HR, we inactivated the nonhomologous end-joining (NHEJ) pathway in UAF1-deficient cells. Disruption of NHEJ in UAF1-deficient cells restored cellular resistance to camptothecin and the PARP inhibitor. Our results indicate that the USP1/UAF1 complex promotes HR, at least in part by suppressing NHEJ.

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA–damage site. Mutations in BRCA1 and BRCA2 predispose hereditary breast and ovarian cancer. Such mutations sensitize to chemotherapeutic agents, including camptothecin, cisplatin, and poly(ADP-ribose) polymerase (PARP) inhibitor, since RAD51 mediators including both BRCA proteins promote repair of DNA lesions induced by these drugs. Little is known of the functional relationships among RAD51, BRCA2, and other RAD51 mediators, because no brca2-null cells were available. Furthermore, the phenotype of sws1 mutants has not been documented. We here disrupted every known RAD51 mediator and analyzed the phenotype of the resulting mutants in both BRCA2-deficient and -proficient backgrounds. The understanding of the function of individual RAD51 mediators and their functional interactions will contribute to the accurate prediction of anti-cancer therapy efficacy.