No abstract

We showed in this study that cells deficient of the BRCA1-associated BACH1 helicase, also known as BRIP1, failed to elicit homologous recombination (HR) after DNA double-stranded breaks (DSBs). BACH1-deficient cells were also sensitive to mitomycin C (MMC) and underwent MMC-induced chromosome instability. Moreover, we identified a homozygous nonsense mutation in BACH1 in a FA-J patient-derived cell line and could not detect BACH1 protein in this cell line. Expression of wild-type BACH1 in this cell line reduced the accumulation of cells at G2/M phases following exposure to DNA crosslinkers, a characteristic of Fanconi anemia (FA) cells. These results support the conclusion that BACH1 is FANCJ.

The pathway determining malignant cellular transformation, which depends upon mutation of the BRCA1 tumor suppressor gene, is poorly defined. A growing body of evidence suggests that promotion of DNA double-strand break repair by homologous recombination (HR) may be the means by which BRCA1 maintains genomic stability, while a role of BRCA1 in error-prone nonhomologous recombination (NHR) processes has just begun to be elucidated. The BRCA1 protein becomes phosphorylated in response to DNA damage, but the effects of phosphorylation on recombinational repair are unknown. In this study, we tested the hypothesis that the BRCA1-mediated regulation of recombination requires the Chk2-and ATM-dependent phosphorylation sites. We studied Rad51-dependent HR and random chromosomal integration of linearized plasmid DNA, a subtype of NHR, which we demonstrate to be dependent on the Mre11-Rad50-Nbs1 complex. Prevention of Chk2-mediated phosphorylation via mutation of the serine 988 residue of BRCA1 disrupted both the BRCA1-dependent promotion of HR and the suppression of NHR. Similar results were obtained when endogenous Chk2 kinase activity was inhibited by expression of a dominant-negative Chk2 mutant. Surprisingly, the opposing regulation of HR and NHR did not require the ATM phosphorylation sites on serines 1423 and 1524. Together, these data suggest a functional link between recombination control and breast cancer predisposition in carriers of Chk2 and BRCA1 germ line mutations. We propose a dual regulatory role for BRCA1 in maintaining genome integrity, whereby BRCA1 phosphorylation status controls the selectivity of repair events dictated by HR and error-prone NHR.The tumor suppressor gene BRCA1 is mutated in up to 50% of cases of familial early-onset breast cancer and in most families with hereditary breast and ovarian cancer (50). BRCA1 is necessary for cellular processes ranging from cell cycle checkpoint control, DNA repair, regulation of transcription, protein ubiquitination, and apoptosis to chromatin remodeling (28,48,54,63). Dysfunction of many or all of these BRCA1 properties may be invoked in cancer development.Of the many types of DNA damage, DNA double-strand breaks (DSBs) represent a particularly dangerous form of damage. If not properly repaired, a DSB causes genetic changes and/or cell death. DSBs can arise spontaneously or may be induced by exogenous DNA damaging agents. The cell utilizes two principal pathways for the repair of DSBs: homologous recombination (HR) and nonhomologous recombination (NHR) (29, 62). Homology-mediated repair requires an undamaged template molecule that contains a homologous DNA sequence ordinarily on a sister chromatid or a homologous chromosome. HR is mediated through multiple proteins, including the Rad51/Rad52 recombinases and BRCA2. Several lines of evidence have indicated a role of BRCA1 in the HR pathway. BRCA1 colocalizes with BRCA2 and Rad51 (10, 41, 52, 70) and forms ionizing radiation (IR)-induced subnuclear foci (IRIF) containing Rad51 protein. Rad51 IRIF are reduc...

The amino acid antiporter system Xc − is important for the synthesis of glutathione (GSH) that functions to prevent lipid peroxidation and protect cells from nonapoptotic, iron-dependent death (i.e., ferroptosis). While the activity of system Xc − often positively correlates with the expression level of its light chain encoded by SLC7A11, inhibition of system Xc − activity by small molecules (e.g., erastin) causes a decrease in the intracellular GSH level, leading to ferroptotic cell death. How system Xc − is regulated during ferroptosis remains largely unknown. Here we report that activating transcription factor 3 (ATF3), a common stress sensor, can promote ferroptosis induced by erastin. ATF3 suppressed system Xc − , depleted intracellular GSH, and thereby promoted lipid peroxidation induced by erastin. ATF3 achieved this activity through binding to the SLC7A11 promoter and repressing SLC7A11 expression in a p53-independent manner. These findings thus add ATF3 to a short list of proteins that can regulate system Xc − and promote ferroptosis repressed by this antiporter.

DNA double stranded breaks (DSBs) are generated by the RAG endonuclease in all developing lymphocytes as they assemble antigen receptor genes1. DNA cleavage by RAG occurs only at the G1-phase of the cell cycle and generates two hairpin-sealed DNA (coding) ends that require nucleolytic opening prior to their repair by classical non-homologous end-joining (NHEJ)1–3. Although there are several cellular nucleases that could perform this function, only the Artemis nuclease is able to do so efficiently2, 3. Here we show, in vivo, that the histone protein H2AX prevents nucleases other than Artemis from processing hairpin-sealed coding ends; in the absence of H2AX, CtIP can efficiently promote the hairpin opening and resection of DNA ends generated by RAG cleavage. This CtIP-mediated resection is inhibited by γ-H2AX and by MDC-1, which binds to γ-H2AX in chromatin flanking DNA DSBs. Moreover, the ATM kinase activates antagonistic pathways that modulate this resection. CtIP DNA end resection activity is normally limited to cells at post-replicative stages of the cell cycle where it is essential for homology-mediated repair4, 5. In G1-phase lymphocytes, DNA ends that are processed by CtIP are not efficiently joined by classical NHEJ and the joints that do form frequently use micro-homologies and exhibit significant chromosomal deletions. Thus, H2AX preserves the structural integrity of broken DNA ends in G1-phase lymphocytes thereby preventing these DNA ends from accessing repair pathways that promote genomic instability.

The cell cycle checkpoint proteins ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its major downstream effector checkpoint kinase 1 (CHK1) prevent the entry of cells with damaged or incompletely replicated DNA into mitosis when the cells are challenged by DNA damaging agents, such as radiation therapy (RT) or chemotherapeutic drugs, that are the major modalities to treat cancer. This regulation is particularly evident in cells with a defective G1 checkpoint, a common feature of cancer cells, due to p53 mutations. In addition, ATR and/or CHK1 suppress replication stress (RS) by inhibiting excess origin firing, particularly in cells with activated oncogenes. Those functions of ATR/CHK1 make them ideal therapeutic targets. ATR/CHK1 inhibitors have been developed and are currently used either as single agents or paired with radiotherapy or a variety of genotoxic chemotherapies in preclinical and clinical studies. Here, we review the status of the development of ATR and CHK1 inhibitors. We also discuss the potential mechanisms by which ATR and CHK1 inhibition induces cell killing in the presence or absence of exogenous DNA damaging agents, such as RT and chemotherapeutic agents. Lastly, we discuss synthetic lethality interactions between the inhibition of ATR/CHK1 and defects in other DNA damage response (DDR) pathways/genes.

No abstract

The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1؉ , rad26 ؉ , and hus1 ؉ ) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S-and G 2 -phase-specific cell killing, delayed kinetics of ␥-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G 2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.