ATP-dependent specific binding of Tn3 transposase to Tn3 inverted repeats

Transposons are discrete segments of DNA which are capable of moving from one site in a genome to many different sites. Tn3 is a prokaryotic transposon which is 4,957 base pairs (bp) long and encodes a transposase protein which is essential for transposition. We report here a simple method for purifying Tn3 transposase and demonstrate that the transposase protein binds specifically to the ends of the Tn3 transposon in an ATP-dependent manner. The transposase protein binds to linear double-stranded DNA both nonspecifically and specifically; the nonspecific DNA binding activity is sensitive to challenge with heparin. Site-specific DNA binding to the ends (inverted repeats) of Tn3 is observed only when binding is performed in the presence of ATP; this ATP-dependent site-specific DNA binding activity is resistant to heparin challenge. Our results indicate that ATP qualitatively alters the DNA binding activity of the transposase protein so that the protein is able to bind specifically to the ends of the Tn3 transposon.     

Transposition without duplication of infecting bacteriophage Mu DNA

Most models of DNA transposition invoke replication of the transposable element, but it is not clear whether a 'co-integrate' is an obligatory intermediate in the pathway leading to the production of simple insertions during transposition. Such an intermediate can be accounted for only by a replicative transposition scheme. Bacteriophage Mu is a temperate phage that can either lysogenize or lyse its host, and it encodes at least two modes of transposition as judged by the end-products generated by the process. During the lytic development of the integrated prophage, co-integrates are the predominant end-products; transposition is coupled to replication during this phase. A small number of simple insertions are also produced during the lytic growth, but during transposition from the infecting phage into the host chromosome, simple insertions are the main end-products. Conditions can be found where the choice between the two kinds of end-products depends on a delicate balance between the essential transposition functions encoded by Mu. Experiments have suggested that the simple insertions which arise during transposition from the infecting phage may do so without Mu DNA replication. Here I demonstrate using an infecting phage with completely methylated DNA, a dam- (DNA adenine methylase) host and a combination of restriction enzymes that can cut either fully methylated or unmethylated DNA but not hemi-methylated DNA, that transposition of the phage DNA into the host chromosome does not involve a duplication of its DNA. This result may also have significance for other transposons that do not appear to go through a co-integrate intermediate during transposition.     

Evidence for a conservative pathway of transposition of bacteriophage Mu

During its lytic cycle bacteriophage Mu uses repeated transposition as a mode of DNA synthesis. These transpositional events are undoubtedly replicative, and presumably semi-conservative. In a Mu lysogen this type of transposition can start immediately after prophage induction. However, in an infective cycle the Mu genome (which is injected into the host cell as a linear molecule flanked by short random sequences of bacterial DNA) must first become integrated into the host chromosome. Little is known about how this occurs apart from the fact that the bacterial sequences at either end of the Mu genome are lost in the process. The integration is thus similar to a transposition event. In an attempt to determine whether this type of Mu transposition (between a linear donor molecule and a circular recipient) is also semi-conservative we have analysed the progeny phage arising from an infective cycle in which the parental DNA was heterozygous for a known genetic marker. The expectation is that if integration of the infecting Mu genome occurs by a single semi-conservative transpositional event then pure phage bursts should be produced as the genetic information on only one strand would be preserved throughout the lytic cycle. The experiments reported here do not support this expectation in that the infected cells yield mixed bursts, suggesting that Mu integration is a conservative, rather than a semi-conservative event.     

DNA sequences at the ends of transposon Tn5 required for transposition

Transposons are a class of genetic elements that can move from one site in a cell's genome to another independently of the cell's general recombination system. Little is known about the mechanism of transposition of compound transposons such as Tn5, but it is thought that a transposon-encoded protein (a transposase) must recognize the outer ends of the element and, together with host factors, catalyse the transfer of the internal DNA into a new site in a manner that may involve replication. It has previously been shown that the synthesis of an IS50R-encoded protein (protein 1) is an essential requirement for Tn5 transposition. Here we demonstrate that a structure containing only the outer 186 base pairs (bp) of both inverted repeats is capable of being efficiently complemented to transpose in Escherichia coli, provided IS50R is located close by on the same replicon. In addition, Bal31-generated deletions indicate that 16-18 bp of the outer end of IS50L are required for transposition. This 16-18-bp sequence contains the 8-9-bp small inverted repeat present at each end of IS50 plus a 9-bp sequence which is homologous to an interrelated sequence present in four copies in the chromosomal origin of replication in a variety of Gram-negative bacteria. This sequence organization suggests that the ends of Tn5 may function to provide a recognition site for the Tn5 transposase adjacent to a sequence recognized by the host replication system.     

Transposition of hAT elements links transposable elements and V(D)J recombination

Transposons are DNA sequences that encode functions that promote their movement to new locations in the genome. If unregulated, such movement could potentially insert additional DNA into genes, thereby disrupting gene expression and compromising an organism's viability. Transposable elements are classified by their transposition mechanisms and by the transposases that mediate their movement. The mechanism of movement of the eukaryotic hAT superfamily elements was previously unknown, but the divergent sequence of hAT transposases from other elements suggested that these elements might use a distinct mechanism. Here we have analysed transposition of the insect hAT element Hermes in vitro. Like other transposons, Hermes excises from DNA via double-strand breaks between the donor-site DNA and the transposon ends, and the newly exposed transposon ends join to the target DNA. Interestingly, the ends of the donor double-strand breaks form hairpin intermediates, as observed during V(D)J recombination, the process which underlies the combinatorial formation of antigen receptor genes. Significant similarities exist in the catalytic amino acids of Hermes transposase, the V(D)J recombinase RAG, and retroviral integrase superfamily transposases, thereby linking the movement of transposable elements and V(D)J recombination.     

Stimulation of protein-directed strand exchange by a DNA helicase

The protein-mediated exchange of strands between a DNA double helix and a homologous DNA single strand involves both synapsis and branch migration, which are two important aspects of any general recombination reaction. Purified DNA-dependent ATPases from Escherichia coli (recA protein), Ustilago (rec 1 protein) and phage T4 (uvsX protein) have been shown to drive both synapsis and branch migration in vitro. The T4 gene 32 protein is a helix-destabilizing protein that greatly stimulates uvsX-protein-catalysed synapsis, and the E. coli SSB (single-strand binding) protein stimulates the analogous recA-protein-mediated reaction to a lesser degree. One suspects that several other proteins also play a role in the strand exchange process. For example, a DNA helicase could in principle accelerate branch migration rates by helping to melt the helix at the branch point. The T4 dda protein is a DNA helicase that is required to move the T4 replication fork past DNA template-bound proteins in vitro. Previously, we have shown that the dda protein binds to a column that contains immobilized T4 uvsX protein. We show here that this helicase specifically stimulates the branch migration reaction that the uvsX protein catalyses as a central part of the genetic recombination process in a T4 bacteriophage-infected cell.     

Importance of DNA stiffness in protein-DNA binding specificity

From the first high-resolution structure of a repressor bound specifically to its DNA recognition sequence it has been shown that the phage 434 repressor protein binds as a dimer to the helix. Tight, local interactions are made at the ends of the binding site, causing the central four base pairs (bp) to become bent and overtwisted. The centre of the operator is not in contact with protein but repressor binding affinity can be reduced at least 50-fold in response to a sequence change there. This observation might be explained should the structure of the intervening DNA segment vary with its sequence, or if DNA at the centre of the operator resists the torsional and bending deformation necessary for complex formation in a sequence dependent fashion. We have considered the second hypothesis by demonstrating that DNA stiffness is sequence dependent. A method is formulated for calculating the stiffness of any particular DNA sequence, and we show that this predicted relationship between sequence and stiffness can explain the repressor binding data in a quantitative manner. We propose that the elastic properties of DNA may be of general importance to an understanding of protein-DNA binding specificity.     

DNA sequence at the end of IS1 required for transposition

The insertion sequence IS1 belongs to a class of bacterial transposable genetic elements that can form compound transposons in which two copies of IS1 flank an otherwise non-transposable segment of DNA. IS1 differs from other known elements of this class (such as IS10, IS50 and IS903) in several respects. It is one of the smallest known insertion elements, exhibits a relatively complex array of open reading frames, is present in the chromosomes of various Enterobacteria, in some cases in many copies, and its insertion can result in the duplication of either 8 or 9 base pairs (bp) in the target DNA. Furthermore, although, like other members of the compound class, it seems to undergo direct transposition, IS1 also promotes replicon fusion (co-integrate formation) at a relatively high frequency. Like all other elements studied to date, the integrity of the extremities of IS1 are essential for efficient transposition. We have constructed a test system to determine the minimal DNA sequences at the extremities of IS1 required for transposition. Sequential deletions of the end sequences reveal that 21-25 bp of an isolated extremity are sufficient for transposition. A specific sequence 13-23 bp from the ends, defining the edge of the minimal sequence, is implicated as an essential site. The sites, symmetrically arrayed at both ends of IS1, correspond to the apparent consensus sequence of the known binding sites for the Escherichia coli DNA-binding protein (called integration host factor or IHF) which is required for the site-specific recombination that leads to integration of bacteriophage lambda into the bacterial genome. The sites at the ends of IS1 may thus bind a host protein, such as JHF or a related protein, that is involved in regulating the transposition apparatus.     

铜绿假单胞菌鞭毛运动相关基因的研究

建立优化的转化条件,将M u转座复合物电转化到临床分离的一株铜绿假单胞菌(P seud om onasaerug inosa)PA 68中,最高转化效率达3.66×104CFU/μg DNA.通过表型筛选,得到三株鞭毛运动能力缺陷的突变子,Sourn thern杂交证实转座子为单点插入.经基因克隆、核苷酸测序研究,证明转座子分别插入到uvrD、phzF 1、zw f三个基因中,这是首次在国际上将M u转座重组技术应用到鞭毛运动相关基因的研究中.由于人工M u转座技术具有随机单点插入的优点,克服了传统转座子能在染色体上迁移的缺点,为进一步研究P.aerug inosa的鞭毛运动机理及致病性奠定基础.     

An Arabidopsis hAT-like transposase is essential for plant development

A significant proportion of the genomes of higher plants and vertebrates consists of transposable elements and their derivatives. Autonomous DNA type transposons encode a transposase that enables them to mobilize to a new chromosomal position in the host genome by a cut-and-paste mechanism. As this is potentially mutagenic, the host limits transposition through epigenetic gene silencing and heterochromatin formation. Here we show that a transposase from Arabidopsis thaliana that we named DAYSLEEPER is essential for normal plant growth; it shares several characteristics with the hAT (hobo, Activator, Tam3) family of transposases. DAYSLEEPER was isolated as a factor binding to a motif (Kubox1) present in the upstream region of the Arabidopsis DNA repair gene Ku70. This motif is also present in the upstream regions of many other plant genes. Plants lacking DAYSLEEPER or strongly overexpressing this gene do not develop in a normal manner. Furthermore, DAYSLEEPER overexpression results in the altered expression of many genes. Our data indicate that transposase-like genes can be essential for plant development and can also regulate global gene expression. Thus, transposases can become domesticated by the host to fulfil important cellular functions.     

Similarity of the Cin1 repetitive family of Zea mays to eukaryotic transposable elements

It has been suggested that the middle repetitive class of sequences that make up a large proportion of the eukaryotic genome have been amplified and dispersed by DNA transposition. Transposition is a phenomenon first postulated by Barbara McClintock on the basis of her genetic analysis of mutants in Zea mays. Since then, DNA transposition has been studied genetically in various plant systems and is well documented on the molecular level in both prokaryotes and eukaryotes. This has included the isolation of DNA inserts at various loci in several plants; however, the prevalence of transposition in plants is not established. We report here DNA nucleotide sequence data which show that some members of the Cin1 middle repetitive family of maize have features characteristic of known transposable elements. One cloned Cin1 repeat has a 6-base pair (bp) perfect inverted repeat sequence at its ends. The terminal five base pairs (5' TGTTG . . . CAACA 3') are identical to the termini of Drosophila copia transposable elements. Two other Cin1 alleles are flanked by 5-bp direct repeats. A comparison is made with the long terminal repeat (LTR) of the copia-Ty1-retrovirus families of moveable genetic elements.     

新编粤剧《目连救母》中佛、儒孝道观的矛盾与融合

中国儒家的孝道观和原始佛教的孝道观存在很大的差异，这些差异给佛、儒孝道观的融合带来了很大阻碍。最终，佛教依靠它的圆融性，儒家也凭借它的包容性，使两种孝道观相互融合。这种矛盾和融合在目连戏中表现得尤为突出。新编粤剧《目连救母》依旧沿袭了目连戏“孝”的主旨，但是这部戏较其他地方剧种对孝的表现力度更大。其中佛、儒孝道观的矛盾和融合的也体现的更加明显。     

A new-specificity mutant of 434 repressor that defines an amino acid-base pair contact

The repressor encoded by bacteriophage 434 binds to its operators by inserting a 'recognition' alpha-helix into the major groove of the DNA. We have identified an amino acid-base pair contact that determines (in part) the DNA-binding specificity of 434 repressor. The identification is based on the properties of a 'new-specificity' mutant, named Repressor [Ala 28], which bears the substitution of Ala for Gln at the first residue of its recognition alpha-helix. Repressor [Ala 28] binds with high affinity to a particular doubly mutant operator bearing the same substitution at position 1 in each half-site, but does not bind to either the wild-type operator or to other mutant operators. We describe molecular models of residue 28-base pair 1 interactions that account for the binding specificities of both the mutant and wild-type proteins.     

H2AX prevents CtIP-mediated DNA end resection and aberrant repair in G1-phase lymphocytes

DNA double-strand breaks (DSBs) are generated by the recombination activating gene (RAG) endonuclease in all developing lymphocytes as they assemble antigen receptor genes. 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 before their repair by classical non-homologous end-joining (NHEJ). Although there are several cellular nucleases that could perform this function, only the Artemis nuclease is able to do so efficiently. Here, in vivo, we show that in murine cells 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 (mediator of DNA damage checkpoint 1), which binds to γ-H2AX in chromatin flanking DNA DSBs. Moreover, the ataxia telangiectasia mutated (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, in which it is essential for homology-mediated repair. 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 show 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.     

姆潘巴效应的实验研究

“姆潘巴效应”被发现40年以来，至今尚无一个完美的答案，本文试图通过实验探索“姆潘巴效应”产生的条件和原因．实验证明，被列为“物理之谜”的“姆潘巴效应”并非是一种违反常规的、奇特的现象，实验揭示，此现象的产生是由于冰箱内温度不均匀所致，是一种很正常的热学现象，简言之，姆潘巴效应不存在．     

Homology-dependent interactions in phage lambda site-specific recombination

General recombination shows a dependence on large regions of homology between the two participating segments of DNA. Many site-specific recombination systems also exhibit a dependence on homology, although in these systems the requirement is limited to a short region (less than 10 base pairs (bp]. We have used the in vitro phage lambda integration reaction to study the role of homology in this model site-specific recombination system. We find that certain non-homologous pairings which are strongly blocked for complete recombination, nevertheless make one pair of strand-exchanges to generate a joint molecule of the Holliday structure type. This result rules out recombination models in which the only homology-dependent step is synapsis (the juxtaposing of the two recombination sites). Our results also reveal a functional asymmetry in the recombination sites. We present models for bacteriophage lambda integrative recombination which accommodate these findings.     

李德裕抑杜牧质疑

许多事实表明,即使是在牛李党争激烈的政治背景下,李德裕抑杜牧也并不存在。会昌年间,杜牧出任外州刺史是仇士良宦官集团在起作用。大中年间,杜牧在文章中流露出对李德裕的不满不仅与当时特定的政治背景有关,同时,与杜牧长期以来对宦官集团的顾忌也不无联系。     

Spatial relationship of the Fis binding sites for Hin recombinational enhancer activity

Site-specific recombination reactions involve the joining or rearrangement of discrete DNA segments in a highly precise manner. A site-specific DNA inversion regulates the expression of flagellin genes in Salmonella by switching the orientation of a promoter. Analysis of the reaction has shown that, in addition to DNA sequences at the two boundaries of the 1-kilobase invertible segment where strand exchange occurs, another cis acting sequence is required for efficient inversion. This 60-base-pair enhancer-like sequence can function at many different locations and in either orientation in a plasmid substrate. It includes two binding sites for a host protein called Factor II or Fis (refs 4 and 5). Here we have investigated the importance of the spatial relationship between the two Fis binding sites for enhancer activity and have found that the correct helical positioning of the binding sites on the DNA is critical. However, this result could not be accounted for by effects on Fis binding. We propose a model for enhancer function in which the enhancer region acts to align the recombination sites into a specific conformation required for productive synapsis.     

Structure of the cro repressor from bacteriophage lambda and its interaction with DNA

The three-dimensional structure of the 66-amino acid cro repressor protein of bacteriophage lambda suggests how it binds to its operator DNA. We propose that a dimer of cro protein is bound to the B-form of DNA with the 2-fold axis of the dimer coincident with the 2-fold axis of DNA. A pair of 2-fold-related alpha-helices of the repressor, lying within successive major grooves of the DNA, seem to be a major determinant in recognition and binding. In addition, the C-terminal residues of the protein, some of which are disordered in the absence of DNA, appear to contribute to the binding.