用DNA酶切小分子片段在非洲爪蟾卵中提取物中实现非细胞体系核装配

用质粒ｐＢＲ３２２的ＤＮＡ酶切片段,长度分别为７５０,３７５和１８６ｂｐ作为外源ＤＮＡ在非洲爪蟾卵撮以物中实现了非细胞体系的核装配。将分离纯化得到的ｐＢＲ３２２ＤＮＡ酶切后经低熔点琼脂糖回收ＤＮＡ片段,长度为７５０,３７５和１８６ｂｐ,分别加入到爪蟾卵提取物再生系统中温育,经ＤＡＰＩ染色,孚尔根染色及电子显微镜观察发现能装配量的重建核。     

应用大肠杆菌染色素DNA在非洲爪蟾卵提取物实现诱导非细胞体系…

近年来我们实验室已成功地利用细胞核体外组装的实验模式,将多种生物的ＤＮＡ在非洲爪蟾卵提取物中实现了非细胞体系核装配。但亲缘关系最远的原核生物的染色体ＤＮＡ是否也能在此真核体系中进行核装配一直没有报道。我们以大肠杆菌染色体ＤＮＡ为材料,研究了它诱导的非细胞体系核装置。在光镜与电镜水平观察了核装配的过程。显微分光光度计扫描显示ＤＮＡ片段在核装配过程中经历了凝集－去凝集的变化。证明大肠杆菌染色体ＤＮＡ也     

应用大肠杆菌染色体DNA在非洲爪蟾卵提取物实现诱导非细胞体系核装配

近年来我们实验室已成功地利用细胞核体外组装的实验模式,将多种生物的DNA在非洲爪蟾卵提取物中实现了非细胞体系核装配。但亲缘关系最远的原核生物的染色体DNA是否也能在此真核体系中进行核装配一直没有报道。我们以大肠杆菌染色体DNA为材料,研究了它诱导的非细胞体系核装配。在光镜与电镜水平观察了核装配的过程。显微分光光度计扫描显示DNA片段在核装配过程中经历了凝集-去凝集的变化。证明大肠杆菌染色体DNA也能诱导爪蟾卵提取物装配成具有典型结构的核。α-~(32)P-dCTP的掺入实验表明重建核具有较高的DNA复制活性。     

诸葛菜去膜精子在爪蟾卵提取物中实现非细胞体系核重建

动物爪蟾(Xenopus laevis)卵提取物诱导植物诸葛菜(Orychophragmus violaceus)去膜精子实现非细胞体系核重建. 诸葛菜去膜精子在爪蟾卵提取物中温育30 min左右开始膨大, 随着温育时间的延长, 膨大的精子染色质逐渐去凝集. 电子显微镜观察和荧光显微镜观察均表明重建核有核膜的装配, 膜泡在去凝集的染色质周围逐渐融合形成双层核膜. 用细胞分级抽提整装电子显微镜技术观察到重建核中有核纤层和核骨架结构的装配.     

甲藻染色体在非细胞体系核重建过程中核小体的组装

利用纯化的原始真核生物寇氏隐甲藻（Crypthecodinium cohnii E.）染色体,使之与非洲爪蟾（Xenopus laevis L.）S期卵提取物温育,发现甲藻染色体经历了一系列去凝集、再凝集的形态变化,最后形成类似典型高等真核生物的间期核结构。小球菌核酸酶酶切分析表明,不具备组蛋白和核小体结构的甲藻染色体在非洲爪蟾卵提取物中进行了核小体装配,此过程与DNA序列本身、核膜以及核纤层蛋白（Lamin）是否存在无关,但部分拓扑异构酶Ⅱ（TopoⅡ）参与了这个过程,说明核小体的组装并非为核重建所必需,决定染色体高级结构的因素并不在DNA本身,而可能是非细胞体系中的组蛋白和非组蛋白。     

四膜虫（Tetrahymena shanghaiensis）大核核仁与去膜大核能诱导非…

外源ＤＮＡ或染色质在非洲爪蟾卵提取物中可以诱导细胞核样结构的重建。重建核除不具有核仁样结构外，在其它形态结构上与真核细胞核十分相似。前人的工作 重建中具有核仁前体结构。但可以是由于缺洗涤戌一核仁组织者的缘故，这些核仁前体不能相互融合形成新生核仁。那么活性核仁组织 重建核中是否能发挥其功能呢？为了研究这一问题，我们提取纯化了四膜虫的大核与大核的周边核仁。进一步去除大核与大核核仁分别加入非洲爪蟾卵非细     

非细胞体系核重建并非必需核小体与染色质的装配

以质粒DNA在爪蟾卵提取物S- 1 5 0中进行核小体构建时形成的超螺旋结构检测核小体的形成 ;利用阳离子交换剂CM -Cellulose定量结合组蛋白H2A和H2B ;并结合小球菌核酸酶分析核小体的形成 ,研究了爪蟾去膜精子在去除H2A ,H2B的S- 1 5 0中的核重建过程 .结果表明CM -Cellulose可有效去除组蛋白并阻止质粒DNA的核小体构建和精子染色质的改建 .但处理后的S- 1 5 0与膜泡组分仍可诱导去膜精子进行体外核重建 ,进一步表明非细胞体系核重建与外源DNA长度无关 ;核小体及染色质的组装对于核重建并非必需 .     

非细胞体系核重建过程中两类膜泡在环形片层及核被膜形成中的作用

非洲爪蟾卵经钙离子载体Ａ２３１８７激活后,在１０,０００ｇ下离心得到爪蟾卵提取物,ＬａｍｂｄａＤＮＡ中入上述提取物可构建出染色质结构,并在染色质表面重建核被膜,同时,在染色质外的区域形成环形片层。核被膜在环形片层有相似的发生途径,它们都是由两类在形态、大小、膜结构上有明显差别的膜泡融合而来,首先是直径２００ｎｍ的圆形小膜泡相互融合成双层膜片层,同时核孔复合体在双层膜上大量装配;以这些双层膜片层为基     

四膜虫(Tetrahymena shanghaiensis)大核核仁与去膜大核能诱导非细胞体系核重建

外源DNA或染色质在非洲爪蟾卵提取物中可以诱导细胞核样结构的重建。重建核除不具有核仁样结构外,在其它形态结构上与真核细胞核十分相似。前人的工作表明在重建核中具有核仁前体结构。但可能是由于缺少活性核仁组织者的缘故,这些核仁前体不能相互融合形成新生核仁。那么活性核仁组织者在重建核中是否能发挥其功能呢?为了研究这一问题,我们提取纯化了四膜虫的大核与大核的周边核仁。进一步去除大核的核被膜,并将去除核被膜的大核与大核核仁分别加入非洲爪赡卵非细胞体系中。通过电镜超薄切片观察,我们发现无论是与大核染色质相连的周边核仁还是分离纯化的核仁结构在非洲爪赡卵非细胞体系中都不能保持其原有结构特征,而是发生了典型核重建变化,并且在诱导形成的重建核中也看不到核仁样结构。这些结果说明具有活性的核仁组织者在加入非洲爪蟾卵提取物后既不能继续保持其原有的RNA转录功能也不能诱导新的核仁的出现。     

体外细胞核组装中核纤层与核膜的关系

采用非洲爪蟾卵提取物非细胞体系,以外源Lambda DNA诱导细胞核的体外组装,以此实验模式为基础,研究了细胞核体外组装过程中核纤层的组装,结果表明核纤层蛋白参与细胞核的体外组装过程,核内骨架的组装与核纤层的组装在时间上是有序的,核内骨架的组装可能为核纤层的装配提供了先决条件.在非洲爪蟾卵提取物非细胞体系中加入抗核纤层蛋白抗体,抑制核纤层的正常装配过程,核膜组装发生异常.结果提示核纤层的组装与核膜的组装是密切相关的.     

Distributions of Topological States in Circular DNA

A distinctive feature of closed circular DNA molecules is their particular topological state, which cannot be altered by any conformational rearrangement short of breaking at least one strand. This topological constraint opens unique possibilities for experimental studies of the distributions of topological states created in different ways. Primarily, the equilibrium distributions of topological properties are considered in the review. It is described how such distributions can be obtained and measured experimentally, and how they can be computed. Comparison of the calculated and measured equilibrium distributions over the linking number of complementary strands, equilibrium fractions of knots and links formed by circular molecules has provided much valuable information about the properties of the double helix. Study of the steady-state fraction of knots and links created by type II DNA topoisomerases has revealed a surprising property of the enzymes: their ability to reduce these fractions considerably below the equilibrium level.     

Topoisomerase II Regulates the Maintenance of DNA Methylation

The maintenance of DNA methylation in nascent DNA is a critical event for numerous biological processes. Following DNA replication, DNMT1 is the key enzyme that strictly copies the methylation pattern from the parental strand to the nascent DNA. However, the mechanism underlying this highly specific event is not thoroughly understood. In this study, we identified topoisomerase IIα (TopoIIα) as a novel regulator of the maintenance DNA methylation. UHRF1, a protein important for global DNA methylation, interacts with TopoIIα and regulates its localization to hemimethylated DNA. TopoIIα decatenates the hemimethylated DNA following replication, which might facilitate the methylation of the nascent strand by DNMT1. Inhibiting this activity impairs DNA methylation at multiple genomic loci. We have uncovered a novel mechanism during the maintenance of DNA methylation.     

FBH1 Helicase Disrupts RAD51 Filaments in Vitro and Modulates Homologous Recombination in Mammalian Cells

Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.     

Human HEL308 localizes to damaged replication forks and unwinds lagging strand structures

HEL308 is a superfamily II DNA helicase, conserved from archaea through to humans. HEL308 family members were originally isolated by their similarity to the Drosophila melanogaster Mus308 protein, which contributes to the repair of replication-blocking lesions such as DNA interstrand cross-links. Biochemical studies have established that human HEL308 is an ATP-dependent enzyme that unwinds DNA with a 3' to 5' polarity, but little else is know about its mechanism. Here, we show that GFP-tagged HEL308 localizes to replication forks following camptothecin treatment. Moreover, HEL308 colocalizes with two factors involved in the repair of damaged forks by homologous recombination, Rad51 and FANCD2. Purified HEL308 requires a 3' single-stranded DNA region to load and unwind duplex DNA structures. When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A. Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures. Together, our results suggest that one role for HEL308 at sites of blocked replication might be to open up the parental strands to facilitate the loading of subsequent factors required for replication restart.     

Studies on human DNA polymerase epsilon and GINS complex and their role in DNA replication

In eukaryotic cells, DNA replication is carried out by the coordinated action of three DNA polymerases (Pols), Pol α, δ, and ε. In this report, we describe the reconstitution of the human four-subunit Pol ε and characterization of its catalytic properties in comparison with Pol α and Pol δ. Human Pol ε holoenzyme is a monomeric complex containing stoichiometric subunit levels of p261/Pol 2, p59, p17, and p12. We show that the Pol ε p261 N-terminal catalytic domain is solely responsible for its ability to catalyze DNA synthesis. Importantly, human Pol (hPol) ε was found more processive than hPol δ in supporting proliferating cell nuclear antigen-dependent elongation of DNA chains, which is in keeping with proposed roles for hPol ε and hPol δ in the replication of leading and lagging strands, respectively. Furthermore, GINS, a component of the replicative helicase complex that is composed of Sld5, Psf1, Psf2, and Psf3, was shown to interact weakly with all three replicative DNA Pols (α, δ, and ε) and to markedly stimulate the activities of Pol α and Pol ε. In vivo studies indicated that siRNA-targeted depletion of hPol δ and/or hPol ε reduced cell cycle progression and the rate of fork progression. Under the conditions used, we noted that depletion of Pol ε had a more pronounced inhibitory effect on cellular DNA replication than depletion of Pol δ. We suggest that reduction in the level of Pol δ may be less deleterious because of its collision-and-release role in lagging strand synthesis.     

The DDN Catalytic Motif Is Required for Metnase Functions in Non-homologous End Joining (NHEJ) Repair and Replication Restart

Metnase (or SETMAR) arose from a chimeric fusion of the Hsmar1 transposase downstream of a protein methylase in anthropoid primates. Although the Metnase transposase domain has been largely conserved, its catalytic motif (DDN) differs from the DDD motif of related transposases, which may be important for its role as a DNA repair factor and its enzymatic activities. Here, we show that substitution of DDN⁶¹⁰ with either DDD⁶¹⁰ or DDE⁶¹⁰ significantly reduced in vivo functions of Metnase in NHEJ repair and accelerated restart of replication forks. We next tested whether the DDD or DDE mutants cleave single-strand extensions and flaps in partial duplex DNA and pseudo-Tyr structures that mimic stalled replication forks. Neither substrate is cleaved by the DDD or DDE mutant, under the conditions where wild-type Metnase effectively cleaves ssDNA overhangs. We then characterized the ssDNA-binding activity of the Metnase transposase domain and found that the catalytic domain binds ssDNA but not dsDNA, whereas dsDNA binding activity resides in the helix-turn-helix DNA binding domain. Substitution of Asn-610 with either Asp or Glu within the transposase domain significantly reduces ssDNA binding activity. Collectively, our results suggest that a single mutation DDN⁶¹⁰ → DDD⁶¹⁰, which restores the ancestral catalytic site, results in loss of function in Metnase.     

Contribution of the intrinsic curvature to measured DNA persistence length

The persistence length of DNA, a, depends both on the intrinsic curvature of the double helix and on the thermal fluctuations of the angles between adjacent base-pairs. We have evaluated two contributions to the value of a by comparing measured values of a for DNA containing a generic sequence and for an "intrinsically straight" DNA. In each 10 bp segment of the intrinsically straight DNA an initial sequence of five bases is repeated in the sequence of the second five bases, so any bends in the first half of the segment are compensated by bends in the opposite direction in the second half. The value of a for the latter DNA depends, to a good approximation, on thermal fluctuations only; there is no intrinsic curvature. The values of a were obtained from measurements of the cyclization efficiency for short DNA fragments, about 200 bp in length. This method determines the persistence length of DNA with exceptional accuracy, due to the very strong dependence of the cyclization efficiency of short fragments on the value of a. We find that the values of a for the two types of DNA fragment are very close and conclude that the contribution of the intrinsic curvature to a is at least 20 times smaller than the contribution of thermal fluctuations. The relationship between this result and the angles between adjacent base-pairs, which specify the intrinsic curvature, is analyzed.     

A procedure for large-scale isolation of RNA-free plasmid and phage DNA without the use of RNase

A preparative procedure for the large-scale isolation of plasmid DNA without the use of RNAse is described. Crude plasmid DNA is prepared using a standard boiling method. High-molecular-weight RNA is removed by precipitation with LiCl, and low-molecular-weight RNA is removed by sedimentation through high-salt solution. The procedure is inexpensive, rapid, simple, and particularly suitable for processing several large-scale preparations simultaneously. A similar procedure has been developed for preparation of lambda-phage DNA.     

Formation of a stable RuvA protein double tetramer is required for efficient branch migration in vitro and for replication fork reversal in vivo

In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2(KaP), specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2(KaP) is fully active for homologous recombination in vivo. RuvA2(KaP) is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2(KaP) for forks is decreased compared with wild type. Accordingly, RuvA2(KaP) is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2(KaP) is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2(KaP) is defective for stimulation of RuvB activity and stability of HJ·RuvA·RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo.