Disparate contributions of the Fanconi anemia pathway and homologous recombination in preventing spontaneous mutagenesis

Fanconi anemia (FA) is a chromosomal instability disorder in which DNA-damage processing defects are reported for translesion synthesis (TLS), non-homologous end joining (NHEJ) and homologous recombination (HR; both increased and decreased). To reconcile these diverse findings, we compared spontaneous mutagenesis in FA and HR mutants of hamster CHO cells. In the fancg mutant we find a reduced mutation rate accompanied by an increased proportion of deletions within the hprt gene. Moreover, in fancg cells gene amplification at the CAD and dhfr loci is elevated, another manifestation of inappropriate processing of damage during DNA replication. In contrast, the rad51d HR mutant has a greatly elevated rate of hprt mutations, >85% of which are deletions. Our analysis supports the concept that HR faithfully restores broken replication forks, whereas the FA pathway acts more globally to ensure chromosome stability by promoting efficient end joining of replication-derived breaks, as well as TLS and HR

The relative efficiency of homology-directed repair has distinct effects on proper anaphase chromosome separation

Homology-directed repair (HDR) is essential to limit mutagenesis, chromosomal instability (CIN) and tumorigenesis. We have characterized the consequences of HDR deficiency on anaphase, using markers for incomplete chromosome separation: DAPI-bridges and Ultra-fine bridges (UFBs). We show that multiple HDR factors (Rad51, Brca2 and Brca1) are critical for complete chromosome separation during anaphase, while another chromosome break repair pathway, non-homologous end joining, does not affect chromosome segregation. We then examined the consequences of mild versus severe HDR disruption, using two different dominant-negative alleles of the strand exchange factor, Rad51. We show that mild HDR disruption is viable, but causes incomplete chromosome separation, as detected by DAPI-bridges and UFBs, while severe HDR disruption additionally results in multipolar anaphases and loss of clonogenic survival. We suggest that mild HDR disruption favors the proliferation of cells that are prone to CIN due to defective chromosome separation during anaphase, whereas, severe HDR deficiency leads to multipolar divisions that are prohibitive for cell proliferation

FANCD1/BRCA2 Plays Predominant Role in the Repair of DNA Damage Induced by ACNU or TMZ

Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. In this work, the role of the Fanconi anemia (FA) repair pathway for DNA damage induced by ACNU or TMZ was examined. Cultured mouse embryonic fibroblasts were used: FANCA−/−, FANCC−/−, FANCA−/−C−/−, FANCD2−/− cells and their parental cells, and Chinese hamster ovary and lung fibroblast cells were used: FANCD1/BRCA2mt, FANCG−/− and their parental cells. Cell survival was examined after a 3 h ACNU or TMZ treatment by using colony formation assays. All FA repair pathways were involved in ACNU-induced DNA damage. However, FANCG and FANCD1/BRCA2 played notably important roles in the repair of TMZ-induced DNA damage. The most effective molecular target correlating with cellular sensitivity to both ACNU and TMZ was FANCD1/BRCA2. In addition, it was found that FANCD1/BRCA2 small interference RNA efficiently enhanced cellular sensitivity toward ACNU and TMZ in human glioblastoma A172 cells. These findings suggest that the down-regulation of FANCD1/BRCA2 might be an effective strategy to increase cellular chemo-sensitization towards ACNU and TMZ

A Systematic Proteomic Study of Irradiated DNA Repair Deficient Nbn-Mice

BACKGROUND: The NBN gene codes for the protein nibrin, which is involved in the detection and repair of DNA double strand breaks (DSBs). The NBN gene is essential in mammals. METHODOLOGY/PRINCIPAL FINDINGS: We have used a conditional null mutant mouse model in a proteomics approach to identify proteins with modified expression levels after 4 Gy ionizing irradiation in the absence of nibrin in vivo. Altogether, amongst approximately 8,000 resolved proteins, 209 were differentially expressed in homozygous null mutant mice in comparison to control animals. One group of proteins significantly altered in null mutant mice were those involved in oxidative stress and cellular redox homeostasis (p<0.0001). In substantiation of this finding, analysis of Nbn null mutant fibroblasts indicated an increased production of reactive oxygen species following induction of DSBs. CONCLUSIONS/SIGNIFICANCE: In humans, biallelic hypomorphic mutations in NBN lead to Nijmegen breakage syndrome (NBS), an autosomal recessive genetic disease characterised by extreme radiosensitivity coupled with growth retardation, immunoinsufficiency and a very high risk of malignancy. This particularly high cancer risk in NBS may be attributable to the compound effect of a DSB repair defect and oxidative stress

Plasticity of BRCA2 Function in Homologous Recombination: Genetic Interactions of the PALB2 and DNA Binding Domains

The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations

Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions

The BRCA2 tumor suppressor plays an important role in the repair of DNA damage by homologous recombination, also termed homology-directed repair (HDR). Human BRCA2 is 3,418 aa and is composed of several domains. The central part of the protein contains multiple copies of a motif that binds the Rad51 recombinase (the BRC repeat), and the C terminus contains domains that have structural similarity to domains in the ssDNA-binding protein replication protein A (RPA). To gain insight into the role of BRCA2 in the repair of DNA damage, we fused a single (BRC3, BRC4) or multiple BRC motifs to the large RPA subunit. Expression of any of these protein fusions in Brca2 mutant cells substantially improved HDR while suppressing mutagenic repair. A fusion containing a Rad52 ssDNA-binding domain also was active in HDR. Mutations that reduced ssDNA or Rad51 binding impaired the ability of the fusion proteins to function in HDR. The high level of spontaneous chromosomal aberrations in Brca2 mutant cells was largely suppressed by the BRC-RPA fusion proteins, supporting the notion that the primary role of BRCA2 in maintaining genomic integrity is in HDR, specifically to deliver Rad51 to ssDNA. The fusion proteins also restored Rad51 focus formation and cellular survival in response to DNA damaging agents. Because as little as 2% of BRCA2 fused to RPA is sufficient to suppress cellular defects found in Brca2-mutant mammalian cells, these results provide insight into the recently discovered diversity of BRCA2 domain structures in different organisms