Genetic and functional interactions between Mus81–Mms4 and Rad27

The two endonucleases, Rad27 (yeast Fen1) and Dna2, jointly participate in the processing of Okazaki fragments in yeasts. Mus81–Mms4 is a structure-specific endonuclease that can resolve stalled replication forks as well as toxic recombination intermediates. In this study, we show that Mus81–Mms4 can suppress dna2 mutational defects by virtue of its functional and physical interaction with Rad27. Mus81–Mms4 stimulated Rad27 activity significantly, accounting for its ability to restore the growth defects caused by the dna2 mutation. Interestingly, Rad27 stimulated the rate of Mus81–Mms4 catalyzed cleavage of various substrates, including regressed replication fork substrates. The ability of Rad27 to stimulate Mus81–Mms4 did not depend on the catalytic activity of Rad27, but required the C-terminal 64 amino acid fragment of Rad27. This indicates that the stimulation was mediated by a specific protein–protein interaction between the two proteins. Our in vitro data indicate that Mus81–Mms4 and Rad27 act together during DNA replication and resolve various structures that can impede normal DNA replication. This conclusion was further strengthened by the fact that rad27 mus81 or rad27 mms4 double mutants were synergistically lethal. We discuss the significance of the interactions between Rad27, Dna2 and Mus81–Mms4 in context of DNA replication.     

Mec1/Tel1-dependent phosphorylation of Slx4 stimulates Rad1–Rad10-dependent cleavage of non-homologous DNA tails

Budding yeast Slx4 interacts with the Rad1–Rad10 endonuclease that is involved in nucleotide excision repair (NER), homologous recombination (HR) and single-strand annealing (SSA). We previously showed that Slx4 is dispensable for NER but is essential for SSA. Slx4 is phosphorylated by the Mec1 and Tel1 kinases after DNA damage on at least six Ser/Thr residues, and mutation of all six residues to Ala reduces the efficiency of SSA. In this study, we further investigated the role of Slx4 phosphorylation in SSA, specifically in regulating cleavage of 3′ non-homologous (NH) DNA tails by Rad1–Rad10 during SSA and HR. Slx4 became phosphorylated after induction of a single double-strand break (DSB) during SSA and dephosphorylation coincided approximately with completion of repair. Slx4 is recruited to 3′ NH tails during DSB repair, but this does not require phosphorylation of Slx4. However, we identified a specific damage-dependent Mec1/Tel1 site of Slx4 phosphorylation, Thr 113, that is required for efficient cleavage of NH tails by Rad1–Rad10. Consistent with these data, deletion of both Mec1 and Tel1 severely reduces the efficiency of NH DNA tail cleavage during HR. These data show that phosphorylation of Slx4 by Mec1 and Tel1 plays an important role in facilitating NH DNA tail cleavage during HR.     

Repair activities of human 8-oxoguanine DNA glycosylase are stimulated by the interaction with human checkpoint sensor Rad9–Rad1–Hus1 complex

Rad9–Rad1–Hus1 (9–1–1) is a checkpoint protein complex playing roles in DNA damage sensing, cell cycle arrest, DNA repair or apoptosis. Human 8-oxoguanine DNA glycosylase (hOGG1) is the major DNA glycosylase responsible for repairing a specific aberrantly oxidized nucleotide, 7,8-dihydro-8-oxoguanine (8-oxoG). In this study, we identified a novel interaction between hOGG1 and human 9–1–1, and investigated the functional consequences of this interaction. Co-immunoprecipitation assays using transiently transfected HEK293 cells demonstrated an interaction between hOGG1 and the 9–1–1 proteins. Subsequently, GST pull-down assays using bacterially expressed and purified hOGG1-His and GST-fused 9–1–1 subunits (GST-hRad9, GST-hRad1, and GST-hHus1) demonstrated that hOGG1 interacted directly with the individual subunits of the human 9–1–1 complex. In vitro excision assay, which employed a DNA duplex containing an 8-oxoG/C mismatch, showed that hRad9, hRad1, and hHus1 enhanced the 8-oxoG excision and β-elimination activities of hOGG1. In addition, the presence of hRad9, hRad1, and hHus1 enhanced the formation of covalently cross-linked hOGG1–8-oxoG/C duplex complexes, as determined by a trapping assay using NaBH₄. A trimeric human 9–1–1 complex was purified from Escherichia coli cell transformed with hRad9, His-fused hRad1, or His-fused hHus1 expressing vectors. It also showed the similar activity to enhance in vitro hOGG1 glycosylase activity, compared with individual human 9–1–1 subunits. Detection of 8-oxoG in HEK293 cells using flow cytometric and spectrofluorometric analysis revealed that over-expression of hOGG1 or human 9–1–1 reduced the formation of 8-oxoG residues following the H₂O₂ treatment. The highest 8-oxoG reduction was observed in HEK293 cells over-expressing hOGG1 and all the three subunits of human 9–1–1. These indicate that individual human 9–1–1 subunits and human 9–1–1 complex showed almost the same abilities to enhance the in vitro 8-oxoG excision activity of hOGG1, but that the greatest effect to remove 8-oxoG residues in H₂O₂-treated cells was derived from the 9–1–1 complex as a whole.     

Rad51–Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-A^CaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-A^CaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-A^CaCse4 levels at the programmed stall sites of early replicating centromeres.     

Human Rad52 binds and wraps single-stranded DNA and mediates annealing via two hRad52–ssDNA complexes

Rad52 promotes the annealing of complementary strands of DNA bound by replication protein A (RPA) during discrete repair pathways. Here, we used a fluorescence resonance energy transfer (FRET) between two fluorescent dyes incorporated into DNA substrates to probe the mechanism by which human Rad52 (hRad52) interacts with and mediates annealing of ssDNA–hRPA complexes. Human Rad52 bound ssDNA or ssDNA–hRPA complex in two, concentration-dependent modes. At low hRad52 concentrations, ssDNA was wrapped around the circumference of the protein ring, while at higher protein concentrations, ssDNA was stretched between multiple hRad52 rings. Annealing by hRad52 occurred most efficiently when each complementary DNA strand or each ssDNA–hRPA complex was bound by hRad52 in a wrapped configuration, suggesting homology search and annealing occur via two hRad52–ssDNA complexes. In contrast to the wild type protein, hRad52^RQK/AAA and hRad52^1–212 mutants with impaired ability to bind hRPA protein competed with hRPA for binding to ssDNA and failed to counteract hRPA-mediated duplex destabilization highlighting the importance of hRad52-hRPA interactions in promoting efficient DNA annealing.     

TopBP1 and DNA polymerase-α directly recruit the 9-1-1 complex to stalled DNA replication forks

TopBP1 and the Rad9–Rad1–Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR–ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-α to sites of replication stress. Furthermore, we show that pol-α is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-α, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.     

Mutators in SACCHAROMYCES CEREVISIAE: MUT1–1, MUT1–2 and MUT2–1

The purpose of this study was to characterize two mutator stocks of yeast which were induced and selected on the basis of high spontaneous reversion rates of the suppressible "ochre" nonsense allele lys1-1. In the mutator stock VA-3, a single mutation, designated mut1-1, is responsible for the increase in the reversion rate of the ochre alleles lys1-1 and arg4-17. In stock VA-105, there are two separate mutator mutations. Tetrad analysis data showed these two loci are loosely linked. Based on complementation data, one of these mutations is at the same locus as mut1-1 and designated mut1-2. The second mutator of stock VA-105 was designated mut2-1. All three mutators are recessive. Both mut1-1 and mut1-2 give a high mutation rate for ochre nonsense suppressor (SUP) loci, but not for the ochre nonsense alleles. On the contrary, the mutation rates of the ochre alleles are greatly reduced. With the mutant mut2-1 there were mutations at both the lys1-1 site and its suppressors; mut2-1 is as effective as mut1-2 but not as effective as mut1-1 in inducing reversions of a missense mutant, his1-7. Neither mut1-1, mut1-2 nor mut2-1 were effective in inducing reversions of a putative frameshift mutation, hom3-10, or in inducing forward mutations to canavanine resistance.     

Rad3 and Sty1 function in Schizosaccharomyces pombe: an integrated response to DNA damage and environmental stress?

In Schizosaccharomyces pombe , the Ataxia Telangiectasia-mutated (Atm)/Atm and Rad 3 Related (Atr) homologue Rad3 is an essential regulator of the response to DNA damage and stalled replication forks. Rad3 activates the downstream kinases Chk1 and Cds1. These kinases in turn inhibit cell cycle progression by mediating Cdc2 phosphorylation. Studies in both yeast and mammalian cells suggest additional roles for Rad3 in regulating cellular responses to environmental stress. In S. pombe , cellular responses to various environmental stresses are regulated primarily through the stress-activated MAP kinase p38 homologue Sty1. An important function of Sty1 is to drive cells rapidly through mitosis by facilitating the accumulation of Cdc25. Interestingly, Sty1 is activated simultaneously with Rad3 following exposure to UV radiation or ionizing radiation (IR). Similarly, exposure to environmental stresses induces the expression of rad3 ⁺, cds1 ⁺ and other checkpoint regulator genes. It is currently unclear how the pathways regulated by Sty1 and Rad3 and their opposing effects on mitosis are integrated. Recent studies suggest that Sty1 and Rad3 function together to regulate the expression of several stress response genes following exposure to IR. In this review, we discuss current knowledge on the interaction of Rad3/Atm and Sty1/p38 in regulating cellular responses to environmental stress and DNA damage.     

Niemann–Pick C1-Like 1 and cholesterol uptake

Niemann–Pick C1-Like 1 (NPC1L1) is a polytopic transmembrane protein responsible for dietary cholesterol and biliary cholesterol absorption. Consistent with its functions, NPC1L1 distributes on the brush border membrane of enterocytes and the canalicular membrane of hepatocytes in humans. As the molecular target of ezetimibe, a hypocholesterolemic drug, its physiological and pathological significance has been recognized and intensively studied for years. Recently, plenty of new findings reveal the molecular mechanism of NPC1L1's role in cholesterol uptake, which may provide new insights on our understanding of cholesterol absorption. In this review, we summarized recent progress in these studies and proposed a working model, hoping to provide new perspectives on the regulation of cholesterol transport and metabolism.     

CCM1–ICAP-1 complex controls β1 integrin–dependent endothelial contractility and fibronectin remodeling

The endothelial CCM complex regulates blood vessel stability and permeability. Loss-of-function mutations in CCM genes are responsible for human cerebral cavernous malformations (CCMs), which are characterized by clusters of hemorrhagic dilated capillaries composed of endothelium lacking mural cells and altered sub-endothelial extracellular matrix (ECM). Association of the CCM1/2 complex with ICAP-1, an inhibitor of β1 integrin, prompted us to investigate whether the CCM complex interferes with integrin signaling. We demonstrate that CCM1/2 loss resulted in ICAP-1 destabilization, which increased β1 integrin activation and led to increased RhoA-dependent contractility. The resulting abnormal distribution of forces led to aberrant ECM remodeling around lesions of CCM1- and CCM2-deficient mice. ICAP-1–deficient vessels displayed similar defects. We demonstrate that a positive feedback loop between the aberrant ECM and internal cellular tension led to decreased endothelial barrier function. Our data support that up-regulation of β1 integrin activation participates in the progression of CCM lesions by destabilizing intercellular junctions through increased cell contractility and aberrant ECM remodeling.     

Characteristics and application of S1–P1 nucleases in biotechnology and medicine

3′–nucleases/nucleotidases of the S1–P1 family (EC 3.1.30.1) are single–strand–specific or non-specific zinc–dependent phosphoesterases present in plants, fungi, protozoan parasites, and in some bacteria. They participate in a wide variety of biological processes and their current biotechnological applications rely on their single–strand preference, nucleotide non-specificity, a broad range of catalytic conditions and high stability. We summarize the present and potential utilization of these enzymes in biotechnology and medicine in the context of their biochemical and structure–function properties. Explanation of unanswered questions for bacterial and trypanosomatid representatives could facilitate development of emerging applications in medicine.     

细胞周期检控蛋白Rad17

Rad17是细胞应答DNA损伤和复制叉阻滞信号转导过程中一个关键的检控蛋白,在DNA损伤和DNA复制检控中具有非常重要的作用.现对Radl7在DNA损伤检控、DNA复制检控、端粒结构稳定以及减数分裂细胞周期检控中的重要作用进行综述,并探讨Radl7与肿瘤发生的关系.     

Recombination and Chromosome Segregation during the Single Division Meiosis in SPO12–1 and SPO13–1 Diploids

This paper reports a study of chromosome segregation and recombination during sporulation of spo12–1 and spo13–1 diploid strains of S. cerevisiae. These strains undergo a single division to form asci containing two diploid or near-diploid spores. The segregation of centromere-linked markers in the two-spored (dyad) products indicates that the division is generally equational. However, in a small percentage of the spo12–1 and spo13–1 cells, it appears that a meiosis I-like division occurs. Aberrant segregation of the MAT locus on chromosome III, yielding a monosomic and a trisomic spore pair, occurs in 12% of all dyads. The segregation patterns of markers at various distances from their centromeres and several pairs of markers on the same chromosome indicate that recombination takes place in both strains at nearly standard meiotic levels.     

Establishment and characterization of the NCC–SS1–C1 synovial sarcoma cell line

Synovial sarcoma is an aggressive mesenchymal malignancy characterized by unique gene fusions. Tissue culture cells are essential tools for further understanding tumorigenesis and anti-cancer drug development; however, only a limited number of well-characterized synovial sarcoma cell lines exist. Thus, the objective of this study was to establish a patient-derived synovial sarcoma cell line. We established a synovial sarcoma cell line from tumor tissue isolated from a 72-year-old female patient. Prepared cells were analyzed for the presence of gene fusions by fluorescence in situ hybridization, RT-PCR, and karyotyping. In addition, the resulting cell line was characterized by viability, short tandem repeat, colony and spheroid formation, and invasion analyses. Differences in gene enrichment between the primary tumor and cell line were examined by mass spectrometric protein expression profiling and KEGG pathway analysis. Our analyses revealed that the primary tumor and NCC–SS1–C1 cell line harbored the SS18–SSX1 fusion gene typical of synovial sarcoma and similar proteomics profiles. In vitro analyses also confirmed that the established cell line harbored invasive, colony-forming, and spheroid-forming potentials. Moreover, drug screening with chemotherapeutic agents and tyrosine kinase inhibitors revealed that doxorubicin, a subset of tyrosine kinase inhibitors, and several molecular targeting drugs markedly decreased NCC–SS1–C1 cell viability. Results from the present study support that the NCC–SS1–C1 cell line will be an effective tool for sarcoma research.     

Differential accumulation of soluble amyloid β peptides 1–40 and 1–42 in human monocytic and neuroblastoma cell lines

Alzheimer's disease (AD) is characterized by the massive deposition in the brain of the 40-42-residue amyloid beta protein (A(beta)). While A(beta)1-40 predominates in the vascular system, A(beta)1-42 is the major component of the senile plaques in the neuropil. The concentration of both A(beta) species required to form amyloid fibrils in vitro is micromolar, yet soluble A(betas) found in normal and AD brains are in the low nanomolar range. It has been recently proposed that the levels of A(beta) sufficient to trigger amyloidogenesis may be reached intracellularly. To study the internalization and intracellular accumulation of the major isoforms of A(beta), we used THP-1 and IMR-32 neuroblastoma cells as models of human monocytic and/or macrophagic and neuronal lineages, respectively. We tested whether these cells were able to internalize and accumulate 125I-A(beta)1-40 and 125I-A(beta)1-42 differentially when offered at nanomolar concentrations and free of large aggregates, conditions that mimic a prefibrillar stage of A(beta) in AD brain. Our results showed that THP-1 monocytic cells internalized at least 10 times more 125I-A(betas) than IMR-32 neuroblastoma cells, either isolated or in a coculture system. Moreover, 125I-A(beta)1-42 presented a higher adsorption, internalization, and accumulation of undigested peptide inside cells, as opposed to 125I-A(beta)1-40. These results support that A(beta)1-42, the major pathogenic form in AD, may reach supersaturation and generate competent nuclei for amyloid fibril formation intracellularly. In light of the recently reported strong neurotoxicity of soluble, nonfibrillar A(beta)1-42, we propose that intracellular amyloidogenesis in microglia is a protective mechanism that may delay neurodegeneration at early stages of the disease.     

Association between the c.*229C>T polymorphism of the topoisomerase IIβ binding protein 1 (TopBP1) gene and breast cancer

Topoisomerase IIβ binding protein 1 (TopBP1) is involved in cell survival, DNA replication, DNA damage repair and cell cycle checkpoint control. The biological function of TopBP1 and its close relation with BRCA1 prompted us to investigate whether alterations in the TopBP1 gene can influence the risk of breast cancer. The aim of this study was to examine the association between five polymorphisms (rs185903567, rs116645643, rs115160714, rs116195487, and rs112843513) located in the 3′UTR region of the TopBP1 gene and breast cancer risk as well as allele-specific gene expression. Five hundred thirty-four breast cancer patients and 556 population controls were genotyped for these SNPs. Allele-specific TopBP1 mRNA and protein expressions were determined by using real time PCR and western blotting methods, respectively. Only one SNP (rs115160714) showed an association with breast cancer. Compared to homozygous common allele carriers, heterozygous and homozygous for the T variant had significantly increased risk of breast cancer (adjusted odds ratio = 3.81, 95 % confidence interval: 1.63–8.34, p = 0.001). Mean TopBP1 mRNA and protein expression were higher in the individuals with the CT or TT genotype. There was a significant association between the rs115160714 and tumor grade and stage. Most carriers of minor allele had a high grade (G3) tumors classified as T2-T4N1M0. Our study raises a possibility that a genetic variation of TopBP1 may be implicated in the etiology of breast cancer.     

The SbcCD complex of Deinococcus radiodurans contributes to radioresistance and DNA strand break repair in vivo and exhibits Mre11–Rad50 type activity in vitro

Deinococcus radiodurans lacks a homologue of the recB and recC genes, and the sbcA/B genes, of Escherichia coli. Thus, DNA strand break repair in Deinococcus proceeds by pathways that do not utilize these proteins. Unlike E. coli, the absence of recBC and sbcA/sbcB, and presence of only sbcC and sbcD in Deinococcus, indicates an enigmatic role of SbcCD in this bacterium. Studies on sbcCD mutation in Deinococcus showed nearly a 100-fold increase in gamma radiation sensitivity as compared to wild type. The mutant showed a higher rate of in vivo DNA degradation during the post-irradiation recovery period that corresponds to the RecA-dependent DSB repair phase. These cells showed a typical NotI pattern of DNA reassembly during the early phase of DSB repair, but were defective for the subsequent RecA-dependent phase II of DSB repair. Hydrogen peroxide had no effect on cell survival of the mutant. While its tolerance to higher doses of UVC and mitomycin C was significantly decreased as compared to wild type. Purified recombinant SbcCD proteins showed single-stranded endonuclease and 3′ → 5′ double-stranded DNA exonuclease activities similar to that of the Mre11–Rad50 complex, which is required for DNA strand break repair in higher organisms. These results suggested that the Mre11–Rad50 type nuclease activity of SbcCD proteins contributes to the radiation resistance of D. radiodurans perhaps by promoting the RecA-dependent DSB repair required for polyploid genome maturation.     

The α(1–4)(1–6) glucans from sweet and normal corns

After removal of granular starch at low centrifugal force, the centrifugation, at increasing forces, of aqueous extracts of su₁ corn gave a series of α-glucan precipitates that contained amylose. The amylose content decreased as the force increased. In contrast, in normal corn all the α-glucan precipitated as starch granules at low forces. In the sweet corn precipitates, apart from the granular starch, the branched α-glucan was phytoglycogen. The MW of this decreased as the proportion of amylose decreased. It appears that, as well as starch granules and soluble phytoglycogen, sweet corn contains granules, smaller than starch, of a range of sizes, and these are made up of phytoglycogen and amylose. As granule size decreases, so does the MW of the phytoglycogen and the content of amylose. A method of quantitative extraction of starch giving minimal depolymerization is described. The isopotential iodine absorption of a quantitative extract of sweet corn flour indicated that the total ratio of linear (amylose) fraction to branched (amylopectin + phytoglycogen) fraction was near the normal value of 1:4.