Association of crossover points with topoisomerase I cleavage sites: a model for nonhomologous recombination

Nonhomologous DNA recombination is frequently observed in somatic cells upon the introduction of DNA into cells or in chromosomal events involving sequences already stably carried by the genome. In this report, the DNA sequences at the crossover points for excision of SV40 from chromosomes were shown to be associated with eukaryotic topoisomerase I cleavage sites in vitro. The precise location of the cleavage sites relative to the crossover points has suggested a general model for nonhomologous recombination mediated by topoisomerase I.     

DNA looping generated by DNA bending protein IHF and the two domains of lambda integrase

The multiprotein-DNA complexes that participate in bacteriophage lambda site-specific recombination were used to study the combined effect of protein-induced bending and protein-mediated looping of DNA. The protein integrase (Int) is a monomer with two autonomous DNA binding domains of different sequence specificity. Stimulation of Int binding and cleavage at the low affinity core-type DNA sites required interactions with the high affinity arm-type sites and depended on simultaneous binding of the sequence-specific DNA bending protein IHF (integration host factor). The bivalent DNA binding protein is positioned at high affinity sites and directed, by a DNA bending protein, to interactions with distant lower affinity sites. Assembly of this complex is independent of protein-protein interactions.     

Drosophila BLM in double-strand break repair by synthesis-dependent strand annealing

Bloom syndrome, characterized by a predisposition to cancer, is caused by mutation of the RecQ DNA helicase gene BLM. The precise function of BLM remains unclear. Previous research suggested that Drosophila BLM functions in the repair of DNA double-strand breaks. Most double-strand breaks in flies are repaired by homologous recombination through the synthesis-dependent strand-annealing pathway. Here, we demonstrate that Drosophila BLM mutants are severely impaired in their ability to carry out repair DNA synthesis during synthesis-dependent strand annealing. Consequently, repair in the mutants is completed by error-prone pathways that create large deletions. These results suggest a model in which BLM maintains genomic stability by promoting efficient repair DNA synthesis and thereby prevents double-strand break repair by less precise pathways.     

A novel nucleoprotein complex at a replication origin

The viral protein p6, required for the protein-primed initiation of replication of Bacillus subtilis phage phi 29, forms a nucleoprotein complex at the viral replication origins that shows novel features. Deoxyribonuclease I and hydroxyl radical footprinting data, as well as the induction of positive supercoiling, support a model in which a DNA right-handed superhelix tightly wraps around a multimeric p6 core. The interaction occurs through the DNA minor groove. The activity of p6 not only requires the formation of the complex but also its correct positioning, indicating that the other proteins involved in the initiation of replication recognize, at a precise position, either the p6 core or the DNA conformational change induced by p6.     

The Hin invertasome: protein-mediated joining of distant recombination sites at the enhancer

The Hin protein binds to two cis-acting recombination sites and catalyzes a site-specific DNA inversion reaction that regulates the expression of flagellin genes in Salmonella. In addition to the Hin protein and the two recombination sites that flank the invertible segment, a third cis-acting recombinational enhancer sequence and the Fis protein, which binds to two sites within the enhancer, are required for efficient recombination. Intermediates of this reaction were trapped during DNA strand cleavage and analyzed by gel electrophoresis and electron microscopy in order to determine their structure and composition. The analyses demonstrate that the recombination sites are assembled at the enhancer into a complex nucleo-protein structure (termed the invertasome) with the looping of the three segments of intervening DNA. Antibody studies indicated that Fis physically interacts with Hin and that both proteins are intimately associated with the invertasome. In order to achieve this protein-protein interaction and assemble the invertasome, the substrate DNA must be supercoiled.     

利用重叠延伸PCR技术进行DNA的人工合成

重叠延伸PCR技术是一种通过寡聚核苷酸链之间重叠的部分互相搭桥、互为模板进行PCR扩增,从而获得目的DNA基因片段的方法。该技术在目的基因的人工合成及外源蛋白在宿主中的高效表达等方面显示出良好的应用前景。综述了重叠延伸PCR技术的原理、反应条件的优化及其应用。     

Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes

The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.     

Heterogeneity of normal human diploid fibroblasts: isolation and characterization of one phenotype

Cultures of human diploid fibroblasts contain cells that respond to exposure to the first component of complement (C1) by initiating DNA synthesis and growth. The plasma membranes of these cells have specific binding sites for the C1q subcomponent of C1. A fluorescence-activated cell sorter was used to isolate a subset of cells with a high affinity for C1q, and the growth and synthesis activities of these high-affinity cells were studied after numerous replications in vitro. These cells synthesize DNA and grow faster than the parent cultures and low-affinity cells, and they produce two to three times as much protein. About 40 percent of their total protein synthesis activity is directed to collagen production, unusually high proportions of collagen types III and V being produced. These properties and the high affinity of the cells for C1q are retained for at least six cell transfers. This phenotype has the properties expected of fibroblasts in healing wounds and inflamed tissues.     

Metals and DNA: molecular left-handed complements

Chiral metal complexes provide unique molecular probes for DNA. Chiral reagents that "recognize" different local structures along the DNA strand have been designed by a process in which the asymmetry in shape and size of the complex is matched to that of the DNA helical groove. As a result, the chiral metal complexes provide very sensitive probes for local helical structure, both left- and right-handed. Direct coordination of chiral complexes to the DNA bases adds an element of sequence selectivity to the probe design. With a suitable reactive metal center, reagents that target chemically specific sites along the strand may be developed. One such chiral reagent, which cleaves left-handed DNA sites with photoactivation, has been useful in mapping this distinct conformation and examining its biological role. The conformation-specific molecular cleaver, much like a DNA-binding enzyme, recognizes and reacts at discrete sites along the DNA strand. These site-specific chiral metal complexes provide exciting new tools for probing the local variations in DNA structure and its role in the regulation of gene expression.     

油菜脂肪酸延长酶基因fae1片段的克隆与SNP分析

 脂肪酸延长酶基因fae1是调控油菜芥酸合成的关键基因。本研究利用GenBank中的fae1基因序列AF490462为模板设计引物，通过多聚酶链式反应（PCR）从白菜、甘蓝和甘蓝型油菜（包括2个人工合成种）的12个不同品种中扩增出长1 007bp的fae1基因片段。PCR产物经与克隆载体pGEM-T vector连接和序列测定，获得12个品种的fae1基因片段的DNA 序列。对来自12个不同品种的fae1基因序列进行比较分析表明：fae1基因具有高度序列保守性，扩增长度均为1 007 bp，核苷酸序列相似度达98%以上，氨基酸序列的保守性更高。在1007 bp的区间内共发现23个SNP(single nucleotide polymorphism)位点，其中有11个单核苷酸变异导致了编码氨基酸的改变。人工甘蓝型油菜和天然甘蓝型油菜的fae1基因片段未发现明显差异。发现了高芥酸品种与低芥酸品种的fae1基因、以及A基因组与C基因组的fae1基因特有SNP位点。     

Structure and flexibility adaptation in nonspecific and specific protein-DNA complexes

Interaction of regulatory DNA binding proteins with their target sites is usually preceded by binding to nonspecific DNA. This speeds up the search for the target site by several orders of magnitude. We report the solution structure and dynamics of the complex of a dimeric lac repressor DNA binding domain with nonspecific DNA. The same set of residues can switch roles from a purely electrostatic interaction with the DNA backbone in the nonspecific complex to a highly specific binding mode with the base pairs of the cognate operator sequence. The protein-DNA interface of the nonspecific complex is flexible on biologically relevant time scales that may assist in the rapid and efficient finding of the target site.     

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.     

Direct observation of molecular preorganization for chirality transfer on a catalyst surface

The chemisorption of specific optically active compounds on metal surfaces can create catalytically active chirality transfer sites. However, the mechanism through which these sites bias the stereoselectivity of reactions (typically hydrogenations) is generally assumed to be so complex that continued progress in the area is uncertain. We show that the investigation of heterogeneous asymmetric induction with single-site resolution sufficient to distinguish stereochemical conformations at the submolecular level is finally accessible. A combination of scanning tunneling microscopy and density functional theory calculations reveals the stereodirecting forces governing preorganization into precise chiral modifier-substrate bimolecular surface complexes. The study shows that the chiral modifier induces prochiral switching on the surface and that different prochiral ratios prevail at different submolecular binding sites on the modifier at the reaction temperature.     

Multienzyme systems of DNA replication

Replication is accomplished by multienzyme systems whose operations are usefully considered in respect to three stages of the process: initiation, elongation, anid termination. 1) Initiation entails synthesis of a short RNA fragment that serves as primer for the elongation step of DNA synthesis. This stage, probed by SS phage DNA templates, reveals three distinctive and highly specific systems in E. coli. The Ml3 DNA utilizes RNA polymerase in a manner that may reflect how plasmid elements are replicated in the cell. The ?X174 DNA does not rely on RNA-polymerase, but requires instead five distinctive proteins which may belong to an apparatus for initiating a host chromosome replication cycle at the origin. The G4 DNA, also independent of RNA polymerase, needs simply the dnaG protein for its distinctive initiation and may thus resemble the system that initiates the replication fragments at the nascent growing fork. In each case it is essential that in vitro the DNA-unwinding protein coat the viral DNA and influence its structure. 2) Elongation is achieved in every case by the multisubunit, holoenzyme form of DNA polymerase III. Copolymerase III, which is an enzyme subunit, and adenosine triphosphate are required to form a proper complex with the primer template but appear dispensable for the ensuing chain growth by DNA polymerase (33). 3) Termination requires excision of the RNA priming fragment, filling of gaps and sealing of interruptions to produce a covalently intact phosphodiester backbone. DNA polymerase I has the capacity for excision and gapfilling and DNA ligase is required for sealing. What once appeared to be a simple DNA polymerase-mediated conversion of a single-strand to a duplex circle (34) is now seen as a complex series of events in which diverse multienzyme systems function. Annoyance with the difficulties in resolving and reconstituting these systems is tempered by the conviction that these are the very systems used ,by the cell in replicating its chromosome and extrachromosomal elements. Thus, understanding of the regulation of replication events in the cell, their localization at membrane surfaces and integration with cell division, and their coordination with phage DNA maturation and particle assembly will all be advanced by knowledge of the components of the replicative machinery.     

咖啡因Na_2─EDTA对辐照大豆和油菜幼苗DNA合成的影响

本文研究了咖啡固和Ｎａ２一ＥＤＴＡ后处理对不同辐照敏感性作物幼苗ＤＮＡ、ＲＮＡ含量和修复能力的影响。结果表明，随着辐照剂量的增大，大豆（对辐射敏感）和油菜（抗辐射）７天龄幼苗下胚轴内ＤＮＡ，ＲＮＡ含量随之增大，且大豆的ＤＮＡ含量比油菜高２倍多。咖啡固和Ｎａ２一ＥＤＴＡ后处理，均高于对照。放射性３Ｈ─ＴｄＲ标记６渗入法证实，种子辐照后，７日龄幼苗体内仍存在着非按期的ＤＮＡ合成，且油菜的非按期ＤＮＡ合成能力强于大豆。咖啡固、Ｎａ２─ＥＤＴＡ和二者复合作用的后处理，抑制了ＤＮＡ的非按期修复合成，加重了辐射损伤，其抑制程度，咖啡因＋Ｎ８２－ＥＤＴＡ＞咖啡固＞Ｎ８２─ＥＤＴＡ。