Poly(ADP-Ribose) Mediates the BRCA2-Dependent Early DNA Damage Response

Breast cancer susceptibility gene 2 (BRCA2) plays a key role in DNA damage repair for maintaining genomic stability. Previous studies have shown that BRCA2 contains three tandem oligonucleotide/oligosaccharide binding folds (OB-folds) that are involved in DNA binding during DNA double-strand break repair. However, the molecular mechanism of BRCA2 in DNA damage repair remains elusive. Unexpectedly, we found that the OB-folds of BRCA2 recognize poly(ADP-ribose) (PAR) and mediate the fast recruitment of BRCA2 to DNA lesions, which is suppressed by PARP inhibitor treatment. Cancer-associated mutations in the OB-folds of BRCA2 disrupt the interaction with PAR and abolish the fast relocation of BRCA2 to DNA lesions. The quickly recruited BRCA2 is important for the early recruitment of exonuclease 1(EXO1) and is involved in DNA end resection, the first step of homologous recombination (HR). Thus, these findings uncover a molecular mechanism by which BRCA2 participates in DNA damage repair

Poly(ADP-ribosyl)ation is recognized by ECT2 during mitosis

<div><p>Poly(ADP-ribosyl)ation is an unique posttranslational modification and required for spindle assembly and function during mitosis. However, the molecular mechanism of poly(ADP-ribose) (PAR) in mitosis remains elusive. Here, we show the evidence that PAR is recognized by ECT2, a key guanine nucleotide exchange factor in mitosis. The BRCT domain of ECT2 directly binds to PAR both <i>in vitro</i> and <i>in vivo</i>. We further found that α-tubulin is PARylated during mitosis. PARylation of α-tubulin is recognized by ECT2 and recruits ECT2 to mitotic spindle for completing mitosis. Taken together, our study reveals a novel mechanism by which PAR regulates mitosis.</p></div

Loss of BRCA1-A Complex Function in RAP80 Null Tumor Cells

<div><p>Receptor Associated Protein 80 (RAP80) is a subunit of the BRCA1-A complex and targets BRCA1 to DNA damage sites in response to DNA double strand breaks. Since mutations of BRCA1 are associated with familial ovarian cancers, we screened 26 ovarian cancer-derived cell lines for RAP80 mutations and found that TOV-21G cells harbor a RAP80 mutation (c.1107G >A). This mutation generates a stop codon at Trp369, which deletes the partial AIR region and the C-terminal zinc fingers of RAP80. Interestingly, both the mutant and wild type alleles of RAP80 lose their expression due to promoter hypermethylation, suggesting that TOV-21G is a RAP80-null cell line. In these cells, not only is the BRCA1-A complex disrupted, but the relocation of the remaining subunits in the BRCA1-A complex including BRCA1, CCDC98, NBA1, BRCC36 and BRE is significantly suppressed. Moreover, TOV-21G cells are hypersensitive to ionizing radiation, which is due to the compromised DNA damage repair capacity in these cells. Reconstitution of TOV-21G cells with wild type RAP80 rescues these cellular defects in response to DNA damage. Thus, our results demonstrate that RAP80 is a scaffold protein in the BRCA1-A complex. Identification of TOV-21G as a RAP80 null tumor cell line will be very useful for the study of the molecular mechanism in DNA damage response.</p> </div

Loss of RAP80 suppresses the IRIF of the BRCA1-A complex.

<p>(A) The IRIF of BRCA1 is suppressed in TOV-21G cells. HBL100 and TOV-21G cells were treated with 10 Gy of IR. Cells were fixed and examined by indicated antibodies. Bar: 10 µm. (B) The IRIF of the BRCA1-A complex is impaired when loss of RAP80. The IRIF of endogenous CCDC98 and NBA1 was examined by indicated antibodies. The IRIF of BRCC36 and BRE was examined using cells stably expressing Flag-tagged BRCC36 and BRE. Bar: 10 µm. (C) Foci positive cells are summarized. Results are averaged (±s.d.) from three independent experiments. *<i>P</i> values<0.01. (D) The BRCA1-A complex is dissembled in TOV-21G cells. The interaction between endogenous BRCA1 and NBA1 and interaction between CCDC98 and NBA1 were examined by IP and Western blotting using indicated antibodies.</p

Loss of RAP80 abrogates DNA damage repair.

<p>(A) Expression of exogenous RAP80 in TOV-21G cells. (B) Cells loss of RAP80 are hypersensitive to IR. TOV-21G and TOV-21G-RAP80 cells were treated with indicated dose of IR. Survival cell colonies were calculated. (C) Comet assays show that loss of RAP80 impairs DNA damage repair. Representative images of neutral comet assays are shown. Bar: 10 µm. (D) The moment of comet tail were quantitatively measured. *<i>P</i> values<0.01; NS means no statistical significance. (E) Exogenous RAP80 restores the IRIF of the BRCA1-A complex in TOV-21G cells. Bar: 10 µm. (F) Foci positive cells are summarized. Data are from three independent experiments and error bars stand for standard deviation. *<i>P</i> values<0.01.</p

New constraints on the genesis and geodynamic setting of the Wulong gold deposit, Liaodong Peninsula, northeast China: evidence from geology, geochemistry, fluid inclusions, and C–H–O–S–Pb isotopes

The Wulong lode gold deposit is located in the Liaoning Province, northeast part of North China Craton. Gold orebodies are mainly hosted in the Late Jurassic granite and structurally controlled by the NE-trending faults. Mineralization can be divided into three stages: (1) quartz-pyrite stage, (2) quartz-polymetallic sulfides stage, and (3) quartz-carbonate stage. Gold formed mainly in the middle stage. Quartz formed in two earlier stages contains three compositional types of fluid inclusions, i.e. pure CO2, CO2–H2O and NaCl–H2O, but the late-stage minerals only contain the NaCl–H2O inclusions. The inclusions in quartz formed in the early, main and late stages yield total homogenization temperatures of 317–383 °C, 260–380 °C and 159–234 °C, respectively, with salinities of 5.14-9.44, 2.95-6.20, 1.23-4.34 wt% NaCl equivalent, respectively. Trapping pressures estimated from CO2–H2O inclusions are 200–390 MPa in the main stage. Fluid boiling and immiscibility caused rapid precipitation of sulfides and gold. Through immiscibility and inflow of meteoric water, the ore-forming fluid system evolved from CO2-rich to CO2-poor in composition, and from magmatic to meteoric, as indicated by δ18Owater values (4.5‰–7.3‰). The carbon (-12.2‰ to -11.5‰), sulfur (0.9‰–2.6‰) and lead isotope (207Pb/204Pb of 15.606–15.618) compositions suggest the hostrocks to be a significant source of ore metals. Integrating the data obtained from the studies including ore geology, fluid inclusion and isotope geochemistry, we conclude that the Wulong deposit is a decratonization gold deposit formed during lithospheric thinning associated with destruction of the NCC triggered by the subduction of the Paleo-Pacific Oceanic plate in the Early Cretaceous.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

TOV-21G is a RAP80-null cell line.

<p>(A) The expression of RAP80 in 293T, HBL100 and TOV-21G cells was examined by Western blotting. β-actin was used as the protein loading control. (B) The transcription of <i>RAP80</i> gene was examined by RT-PCR. (C) Q-PCR shows the different mRNA level of RAP80 in HBL100 and TOV-21G cells. Results are shown as plus standard deviation (s.d.) from three independent experiments. *<i>P</i> values<0.01. (D) DNA methylation status of the CpG islands at the <i>RAP80</i> promoter region was examined by bisulfit sequencing. 5-AZA treatment significantly decreased the methylation level at the <i>RAP80</i> promoter in TOV-21G cells. (E, F) 5-AZA treatment increases the transcription of <i>RAP80</i> gene in TOV-21G cells. *<i>P</i> values<0.01. (G) 5-AZA treatment induces the protein expression of RAP80 in TOV-21G cells. The wild type and truncated mutant of RAP80 are indicated. β-actin was used as the protein loading control.</p