Regulation of replication fork progression through histone supply and demand

DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize a complex in which the human histone chaperone Asf1 and MCM2-7, the putative replicative helicase, are connected through a histone H3-H4 bridge. Depletion of Asf1 by RNA interference impedes DNA unwinding at replication sites, and similar defects arise from overproduction of new histone H3-H4 that compromises Asf1 function. These data link Asf1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork progression and histone supply and demand.     

DNA damage-induced replication fork regression and processing in Escherichia coli

DNA lesions that block replication are a primary cause of rearrangements, mutations, and lethality in all cells. After ultraviolet (UV)-induced DNA damage in Escherichia coli, replication recovery requires RecA and several other recF pathway proteins. To characterize the mechanism by which lesion-blocked replication forks recover, we used two-dimensional agarose gel electrophoresis to show that replication-blocking DNA lesions induce a transient reversal of the replication fork in vivo. The reversed replication fork intermediate is stabilized by RecA and RecF and is degraded by the RecQ-RecJ helicase-nuclease when these proteins are absent. We propose that fork regression allows repair enzymes to gain access to the replication-blocking lesion, allowing processive replication to resume once the blocking lesion is removed.     

ATR homolog Mec1 promotes fork progression,thus averting breaks in replication slow zones

Budding yeast Mec1, homolog of mammalian ATR, is an essential protein that mediates S-phase checkpoint responses and meiotic recombination. Elimination of Mec1 function leads to genomewide fork stalling followed by chromosome breakage. Breaks do not result from stochastic collapse of stalled forks or other incidental lesions; instead, they occur in specific regions of the genome during a G2 chromosomal transition. Break regions are found to be genetically encoded replication slow zones (RSZs), a newly discovered yeast chromosomal determinant. Thus, Mec1 has important functions in normal S phase and the genome instability of mec1 (and, analogously, ATR-/-) mutants stems from defects in these basic roles.     

Resolution of sister telomere association is required for progression through mitosis

Cohesins keep sister chromatids associated from the time of their replication in S phase until the onset of anaphase. In vertebrate cells, two distinct pathways dissociate cohesins, one acts on chromosome arms and the other on centromeres. Here, we describe a third pathway that acts on telomeres. Knockdown of tankyrase 1, a telomeric poly(ADP-ribose) polymerase caused mitotic arrest. Chromosomes aligned normally on the metaphase plate but were unable to segregate. Sister chromatids separated at centromeres and arms but remained associated at telomeres, apparently through proteinaceous bridges. Thus, telomeres may require a unique tankyrase 1-dependent mechanism for sister chromatid resolution before anaphase.     

纤维素降解菌K7-2产纤维素酶培养条件的优化

对新筛选出的产纤维素酶菌株K7—2进行了产酶培养条件的优化。结果表明．菌株K7—2的最优产酶条件为培养液初始pH值7．0、培养温度34℃、摇瓶转速150r·min^-1、培养时间60h、接种量3．5％：最适碳源是麸皮,最适氮源是黄豆粉。为进一步改造、构建更适合生产纤维素酶的工程菌株开辟了新的途径。     

DNA polymerase required for rapid repair of x-ray--induced DNA strand breaks in vivo

A much higher yield of DNA single-strand breaks was obtained in the DNA polymerase-deficient mutant Escherichia coli K-12 pol A1 after a given dose of x-rays than had been found before in Escherichia coli. The increased yield of single-strand breaks was due to the absence of a rapid repair system, which had not been described in Escherichia coli K-12. This absence probably accounts for the x-ray sensitivity of the pol A1 mutant. The rapid repair system can be reversibly inhibited in pol+ cells.     

Direct evidence for DNA bending at the lambda replication origin

Replication initiation in bacteriophage lambda appears to require wrapping of origin DNA on an approximately 50 angstrom radius in or around the complex with the initiator protein O. Since short lengths of DNA are not that flexible, it may be that runs of coherently spaced deoxyadenylate residues constitute bend sites in the ori sequence that facilitate the process. Earlier data showed that ori DNA has electrophoretic anomalies characteristic of bend sites and that these are augmented by initiator protein binding. Here origin bending is examined by direct measurement of the ability of polymerized ori sequences to form small circles. The smallest circles observed (84 residues) are compatible with the required radius of curvature. Bend sites within the O protein binding sites, bend sites in the spacers between them, plus the inherent flexibility of non-bent DNA in the origin may all contribute to origin bending. The data also show that a bend site is required for O protein binding to DNA.     

Pol kappa: A DNA polymerase required for sister chromatid cohesion

Establishment of cohesion between sister chromatids is coupled to replication fork passage through an unknown mechanism. Here we report that TRF4, an evolutionarily conserved gene necessary for chromosome segregation, encodes a DNA polymerase with beta-polymerase-like properties. A double mutant in the redundant homologs, TRF4 and TRF5, is unable to complete S phase, whereas a trf4 single mutant completes a presumably defective S phase that results in a failure of cohesion between the replicated sister chromatids. This suggests that TRFs are a key link in the coordination between DNA replication and sister chromatid cohesion. Trf4 and Trf5 represent the fourth class of essential nuclear DNA polymerases (designated DNA polymerase kappa) in Saccharomyces cerevisiae and probably in all eukaryotes.     

MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity

Instability of the nuclear genome is a hallmark of cancer and aging. MMS19 protein has been linked to maintenance of genomic integrity, but the molecular basis of this connection is unknown. Here, we identify MMS19 as a member of the cytosolic iron-sulfur protein assembly (CIA) machinery. MMS19 functions as part of the CIA targeting complex that specifically interacts with and facilitates iron-sulfur cluster insertion into apoproteins involved in methionine biosynthesis, DNA replication, DNA repair, and telomere maintenance. MMS19 thus serves as an adapter between early-acting CIA components and a subset of cellular iron-sulfur proteins. The function of MMS19 in the maturation of crucial components of DNA metabolism may explain the sensitivity of MMS19 mutants to DNA damage and the presence of extended telomeres.     

PDGF-induced activation of phospholipase C is not required for induction of DNA synthesis

Platelet-derived growth factor (PDGF) induction of DNA synthesis is believed to involve activation of phospholipase C (PLC) and subsequent accumulation of inositol 1,4,5-triphosphate [I(1,4,5)P3], increase in intracellular Ca2+, activation of protein kinase C (PKC), and receptor down regulation. Generation of these events is triggered by the tyrosine protein kinase (TPK) activity of the PDGF receptor. The TPK inhibitor genistein blocked PDGF induction of these events, including DNA synthesis, with the exception of receptor down regulation. PDGF-induced phosphotyrosine phosphorylations, including receptor autophosphorylation, were inhibited by genistein. Removal of genistein and PDGF resulted in DNA synthesis without the occurrence of PLC activation. These findings indicate that these early events, with the exception of receptor down regulation, are not necessary for PDGF-induced DNA synthesis.     

荧光假单胞菌7-14flhA基因的克隆与功能的初步分析

为了探索flhA基因在荧光假单胞菌7-14(Pf7-14)鞭毛合成的作用,以铜绿假单胞菌UCBPP-PA14的鞭毛蛋白FlhA(NC_008463.1)为诱饵,在荧光假单胞菌Pf-5(NC_004129)、Pf0-1(NC_007492)和SBW25(NC_012660)的伞基因组中进行BLASTP比对分析,结果在Pf-5、Pf0-1和SBW25的全基因组搜索到flhA的同系物,分别命名为flhAPf-5、flhAPf0-1,和flhASBW25.根据三者的核酸序列,设计简并引物flhA-F/flhA-R,以荧光假单胞菌7-14(Pf7-14)基凶组DNA为模板进行PCR扩增,并对得到的片段测序比对,发现该片段2 130 bp为一完整的开放阅读框,命名为flhAPf7-14.flhAPf7-14在氨基酸水平上与flhAPf-5和flhAPf0-1的同源性分别达到93%和94%.Southern杂交证明flhA 在Pf7-14基因组中为单一拷贝.通过同源重组单交换的方式构建了Pf7-14flhA基因插入失活突变株,结果表明flhA突变体缺失了鞭毛装置并丧失了游动性,而互补菌株则恢复了鞭毛合成能力和游动能力.本研究为今后进一步研究Pf7-14鞭毛合成、菌体游动性与该菌定殖能力及生防能力的关系奠定基础.     

Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions

Phosphorylation of the human histone variant H2A.X and H2Av, its homolog in Drosophila melanogaster, occurs rapidly at sites of DNA double-strand breaks. Little is known about the function of this phosphorylation or its removal during DNA repair. Here, we demonstrate that the Drosophila Tip60 (dTip60) chromatin-remodeling complex acetylates nucleosomal phospho-H2Av and exchanges it with an unmodified H2Av. Both the histone acetyltransferase dTip60 as well as the adenosine triphosphatase Domino/p400 catalyze the exchange of phospho-H2Av. Thus, these data reveal a previously unknown mechanism for selective histone exchange that uses the concerted action of two distinct chromatin-remodeling enzymes within the same multiprotein complex.     

Serine-7 of the RNA polymerase II CTD is specifically required for snRNA gene expression

RNA polymerase II (Pol II) transcribes genes that encode proteins and noncoding small nuclear RNAs (snRNAs). The carboxyl-terminal repeat domain (CTD) of the largest subunit of mammalian RNA Pol II, comprising tandem repeats of the heptapeptide consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, is required for expression of both gene types. We show that mutation of serine-7 to alanine causes a specific defect in snRNA gene expression. We also present evidence that phosphorylation of serine-7 facilitates interaction with the snRNA gene-specific Integrator complex. These findings assign a biological function to this amino acid and highlight a gene type-specific requirement for a residue within the CTD heptapeptide, supporting the existence of a CTD code.     

优良玉米自交系昌7-2的特征特性及其利用

昌 7- 2是安阳市农科所 1 983年以山东省昌潍地区农科所 (现潍坊市农科院 )选育的单交种昌单七号 (黄早四×潍 95 )为基础材料 ,经过南北连续自交于 1 986年选育而成。昌 7- 2在类群划分上属于塘四坪头系统 ,它继承了黄早四配合力高 ,雄穗发达的优点 ,克服了其易感拟眼斑病、红叶病和鞘紫斑病的缺点 ,是改良黄早四最成功的自交系。自育成以来 ,特别是安阳农科所以其作父本 ,以 478作母本配成国审品种安玉五号以来 ,已被多家育种单位引进、利用。1　特征特性1 .1　昌 7- 2的植物学特性株高 1 70～ 1 80cm ,穗位高 80cm左右 ,主茎叶片数为 1 9…     

环氧合酶-2的DNA疫苗构建及其在COS-7细胞中表达

构建了环氧合酶-2(Cyclooxygenase-2, COX-2)的DNA疫苗载体pVAX1-COX-2, 并考察在COS-7细胞中表达. 用RT-PCR法从肺癌A549细胞中获得环氧合酶-2基因片段, 此片段插入pVAX1载体构建pVAX1-COX-2真核重组表达质粒, 脂质体介导法转染COS-7细胞, 以pVAX1为对照, 收集稳定转染细胞, 利用RT-PCR和Western-blot分别在mRNA和蛋白水平上考察COX-2表达. 实验结果表明: RT-PCR法从A549细胞mRNA中扩增1.8 kb的COX-2基因片段, 酶切与测序结果表明所构建的真核表达载体pVAX1-COX-2与预期相符, 质脂体法成功地将质粒DNA转染到COS-7细胞中. RT-PCR和Western-blot分析结果表明COX-2已表达. 因此, 所构建的真核表达载体pVAX1-COX-2正确, 且在COS-7细胞中表达.     

BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure

Mutations in the BRCA2 (breast cancer susceptibility gene 2) tumor suppressor lead to chromosomal instability due to defects in the repair of double-strand DNA breaks (DSBs) by homologous recombination, but BRCA2's role in this process has been unclear. Here, we present the 3.1 angstrom crystal structure of a approximately 90-kilodalton BRCA2 domain bound to DSS1, which reveals three oligonucleotide-binding (OB) folds and a helix-turn-helix (HTH) motif. We also (i) demonstrate that this BRCA2 domain binds single-stranded DNA, (ii) present its 3.5 angstrom structure bound to oligo(dT)9, (iii) provide data that implicate the HTH motif in dsDNA binding, and (iv) show that BRCA2 stimulates RAD51-mediated recombination in vitro. These findings establish that BRCA2 functions directly in homologous recombination and provide a structural and biochemical basis for understanding the loss of recombination-mediated DSB repair in BRCA2-associated cancers.     

A small viral RNA is required for in vitro packaging of bacteriophage phi 29 DNA

A small RNA of Bacillus subtilis bacteriophage phi 29 is shown to have a novel and essential role in viral DNA packaging in vitro. This requirement for RNA in the encapsidation of viral DNA provides a new dimension of complexity to the attendant protein-DNA interactions. The RNA is a constituent of the viral precursor shell of the DNA-packaging machine but is not a component of the mature virion. Studies of the sequential interactions involving this RNA molecule are likely to provide new insight into the structural and possible catalytic roles of small RNA molecules. The phi 29 assembly in extracts and phi 29 DNA packaging in the defined in vitro system were strongly inhibited by treatment with the ribonucleases A or T1. However, phage assembly occurred normally in the presence of ribonuclease A that had been treated with a ribonuclease inhibitor. An RNA of approximately 120 nucleotides co-purified with the phi 29 precursor protein shell (prohead), and this particle was the target of ribonuclease action. Removal of RNA from the prohead by ribonuclease rendered it inactive for DNA packaging. By RNA-DNA hybridization analysis, the RNA was shown to originate from a viral DNA segment very near the left end of the genome, the end packaged first during in vitro assembly.