The Fanconi Anemia Pathway in Cancer.

Fanconi anemia (FA) is a complex genetic disorder characterized by bone marrow failure (BMF), congenital defects, inability to repair DNA interstrand cross-links (ICLs), and cancer predisposition. FA presents two seemingly opposite characteristics: (a) massive cell death of the hematopoietic stem and progenitor cell (HSPC) compartment due to extensive genomic instability, leading to BMF, and (b) uncontrolled cell proliferation leading to FA-associated malignancies. The canonical function of the FA proteins is to collaborate with several other DNA repair proteins to eliminate clastogenic (chromosome-breaking) effects of DNA ICLs. Recent discoveries reveal that the FA pathway functions in a critical tumor-suppressor network to preserve genomic integrity by stabilizing replication forks, mitigating replication stress, and regulating cytokinesis. Homozygous germline mutations (biallelic) in 22 FANC genes cause FA, whereas heterozygous germline mutations in some of the FANC genes (monoallelic), such as BRCA1 and BRCA2, do not cause FA but significantly increase cancer susceptibility sporadically in the general population. In this review, we discuss our current understanding of the functions of the FA pathway in the maintenance of genomic stability, and we present an overview of the prevalence and clinical relevance of somatic mutations in FA genes.

DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells.

Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.

The identification of FANCD2 DNA binding domains reveals nuclear localization sequences.

Fanconi anemia (FA) is a recessive genetic disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. The FA pathway consists of at least 21 FANC genes (FANCA-FANCV), and the encoded protein products interact in a common cellular pathway to gain resistance against DNA interstrand crosslinks. After DNA damage, FANCD2 is monoubiquitinated and accumulates on chromatin. FANCD2 plays a central role in the FA pathway, using yet unidentified DNA binding regions. By using synthetic peptide mapping and DNA binding screen by electromobility shift assays, we found that FANCD2 bears two major DNA binding domains predominantly consisting of evolutionary conserved lysine residues. Furthermore, one domain at the N-terminus of FANCD2 bears also nuclear localization sequences for the protein. Mutations in the bifunctional DNA binding/NLS domain lead to a reduction in FANCD2 monoubiquitination and increase in mitomycin C sensitivity. Such phenotypes are not fully rescued by fusion with an heterologous NLS, which enable separation of DNA binding and nuclear import functions within this domain that are necessary for FANCD2 functions. Collectively, our results enlighten the importance of DNA binding and NLS residues in FANCD2 to activate an efficient FA pathway.

Coupling of Homologous Recombination and the Checkpoint by ATR.

ATR is a key regulator of cell-cycle checkpoints and homologous recombination (HR). Paradoxically, ATR inhibits CDKs during checkpoint responses, but CDK activity is required for efficient HR. Here, we show that ATR promotes HR after CDK-driven DNA end resection. ATR stimulates the BRCA1-PALB2 interaction after DNA damage and promotes PALB2 localization to DNA damage sites. ATR enhances BRCA1-PALB2 binding at least in part by inhibiting CDKs. The optimal interaction of BRCA1 and PALB2 requires phosphorylation of PALB2 at S59, an ATR site, and hypo-phosphorylation of S64, a CDK site. The PALB2-S59A/S64E mutant is defective for localization to DNA damage sites and HR, whereas the PALB2-S59E/S64A mutant partially bypasses ATR for its localization. Thus, HR is a biphasic process requiring both high-CDK and low-CDK periods. As exemplified by the regulation of PALB2 by ATR, ATR promotes HR by orchestrating a "CDK-to-ATR switch" post-resection, directly coupling the checkpoint to HR.

Roles for APRIN (PDS5B) in homologous recombination and in ovarian cancer prediction.

APRIN (PDS5 cohesin associated factor B) interacts with both the cohesin complex and the BRCA2 tumor suppressor. How APRIN influences cohesion and DNA repair processes is not well understood. Here, we show that APRIN is recruited to DNA damage sites. We find that APRIN interacts directly with RAD51, PALB2 and BRCA2. APRIN stimulates RAD51-mediated DNA strand invasion. APRIN also binds DNA with an affinity for D-loop structures and single-strand (ss) DNA. APRIN is a new homologous recombination (HR) mediator as it counteracts the RPA inhibitory effect on RAD51 loading to ssDNA. We show that APRIN strongly improves the annealing of complementary-strand DNA and that it can stimulate this process in synergy with BRCA2. Unlike cohesin constituents, its depletion has no impact on class switch recombination, supporting a specific role for this protein in HR. Furthermore, we show that low APRIN expression levels correlate with a better survival in ovarian cancer patients and that APRIN depletion sensitizes cells to the PARP inhibitor Olaparib in xenografted zebrafish. Our findings establish APRIN as an important and specific actor of HR, with cohesin-independent functions.

Breast cancer proteins PALB2 and BRCA2 stimulate polymerase η in recombination-associated DNA synthesis at blocked replication forks.

One envisioned function of homologous recombination (HR) is to find a template for DNA synthesis from the resected 3'-OH molecules that occur during double-strand break (DSB) repair at collapsed replication forks. However, the interplay between DNA synthesis and HR remains poorly understood in higher eukaryotic cells. Here, we reveal functions for the breast cancer proteins BRCA2 and PALB2 at blocked replication forks and show a role for these proteins in stimulating polymerase η (Polη) to initiate DNA synthesis. PALB2, BRCA2, and Polη colocalize at stalled or collapsed replication forks after hydroxyurea treatment. Moreover, PALB2 and BRCA2 interact with Polη and are required to sustain the recruitment of Polη at blocked replication forks. PALB2 and BRCA2 stimulate Polη-dependent DNA synthesis on D loop substrates. We conclude that PALB2 and BRCA2, in addition to their functions in D loop formation, play crucial roles in the initiation of recombination-associated DNA synthesis by Polη-mediated DNA repair.