古菌RecJ蛋白的研究进展

RecJ蛋白属于aspartate-histidine-histidine (DHH)磷酸酯酶超家族,存在于细菌、真核生物和古菌中。细菌RecJ蛋白是一种5′→3′ssDNA外切酶,参与错配修复、同源重组、碱基切除修复等生物学过程。真核生物cell division cycle 45 (Cdc45)蛋白是细菌RecJ核酸酶的同源物,但不具有核酸酶活性。Cdc45蛋白能够与minichromosomemaintenance(MCM)和Go-Ichi-Ni-San(GINS)形成Cdc45-MCM-GINS (CMG)复合物,是真核生物DNA复制的重要组分。在古菌中,几乎所有基因组已测序的古菌均编码一种或多种RecJ蛋白同源物。与细菌RecJ核酸酶不同,古菌RecJ蛋白具有多样化的核酸酶活性,并且能够与MCM和GINS形成类似于真核生物CMG的复合物。因此,古菌RecJ蛋白是参与古菌DNA复制、修复和重组的重要成分。基于目前古菌RecJ蛋白的研究报道,本文综述了古菌RecJ蛋白的活性、结构与功能方面的研究进展,聚焦于不同古菌RecJ蛋白以及它们与细菌RecJ核酸酶和真核生物RecJ同源物的...     

Mcm10在真核细胞复制起始中的作用

真核生物DNA复制需要精确的调控,复制起始顺利开启至关重要,其中复制解旋酶的组装是复制起始的核心。作为解旋酶的核心组件,微小染色体维持蛋白(minichromosome maintenance proteins,Mcms)在真核细胞DNA复制的起始阶段扮演着重要的角色。目前,对Mcm2-7复合物的功能研究已开展较多,其在复制起始中的作用已有较为全面的解释。近些年研究发现,Mcm10也在DNA复制的起始调控中起重要作用。该文对近些年Mcm10在真核细胞复制起始中的功能研究进行综述,以期对相关领域的研究者有所帮助。     

真核细胞染色体DNA复制起始及复制叉稳定性维持的机制

在真核生物中,DNA复制在染色体上特定的多位点起始．当细胞处在晚M及G1期,多个复制起始蛋白依次结合到DNA复制源,组装形成复制前复合体．pre．RC在Gl-S的转折期得到激活,随后,多个直接参与DNA复制又形成的蛋白结合到DNA复制源,启动DNA的复制,形成两个双向的DNA复制又．在染色体上,移动的DNA复制又经常会碰到复制障碍（二级DNA结构、一些蛋白的结合位点、损伤的碱基等）而暂停下来,此时,需要细胞周期检验点的调控来稳定复制叉,否则,会导致复制又垮塌及基因组不稳定．本文就真核细胞染色体DNA复制起始的机制,以及复制又稳定性的维持机制进行简要综述．     

生理条件下的S期检查点

细胞周期检查点在细胞遭遇DNA损伤因子的攻击或遇到营养缺乏等不利因素作用时,能够暂时阻止或减慢细胞周期的进程,是细胞在长期进化中发展起来的抵御DNA损伤的重要机制.不仅如此,最近的研究表明,在正常生理条件下,存在一种S期检查点,对DNA复制的速度进行调控.从分子水平而言,这种调控作用可能是通过一系列细胞周期调控蛋白如ATR、9-1-1复合体、Chk1、Cdc25A和CDK2等的作用来实现的.这种调节作用对细胞至关重要,它使DNA复制速度不致于过快,从而减少复制过程中发生错误的几率,维护基因组的稳定性.     

复制酶体的瞻望和疑问

由若干酶组成的复合体参与双链DNA分子复制过程。这种活性蛋白质复合体称为复制酶体(replitase),而具有这些酶活性的蛋白质并不能独立起作用。这种复合体在DNA复制(S期)开始时才由其各种成分装配而成,而在细胞周期的Gl期则不复存在。在真核细胞的复制酶体中,参与dNTP生物合成的酶和参与DNA复制的蛋白质结合在一起。在此复合体被装配前,细胞内先合成了大量“粘合”蛋白,以便将这些有关的蛋白质粘合在一起。在真核细胞内是否有这样的复制酶体存在是多年来一个有争论的问题。作者认为真核细胞中的复制酶体可能由细胞核内、外的两个蛋白质复合体所组成,并由两者协同完成DNA复制。如果复制酶体的存在得到证实,将会对诸如细胞周期、DNA复制、癌症发生、抗癌化学疗法和抗代谢物的生物学作用等理论,产生相当大的影响。     

真核细胞DNA复制叉稳定性维持机制的研究进展

DNA复制是细胞最基本的生命活动之一,是生物体生存和繁殖的基础。从原核生物到真核生物,DNA复制过程基本保守,分为复制起始和延伸两个阶段。复制叉是DNA复制的基本结构,它容易遭受多种内源或外源的DNA复制压力影响而停顿,导致基因组不稳定,引起细胞凋亡、癌变或细胞死亡等严重后果。为了维持复制叉的稳定,细胞进化出了一系列机制,其中最重要机制之一便是S期细胞周期检验点。就影响DNA复制叉稳定的内外因素、S期细胞周期检验点与复制叉稳定性的关系以及复制叉稳定性与相关疾病的发生、治疗等问题进行简要综述。     

Nature发文揭示真核生物DNA复制解旋酶高分辨三维结构

<正>2015年7月29日,清华大学高宁研究组和香港科技大学戴碧瓘研究组共同在Nature以长文形式在线发表研究论文,首次报道真核生物DNA复制起始与延伸过程中DNA解旋酶核心组分MCM2-7复合物的3.8埃高分辨率冷冻电镜结构,并以此为基础对DNA复制起始时MCM2-7复合物的作用机理进行了分析。     

真核生物DNA复制起始调控

复制起始调控是真核生物复制调控机制的重要环节,也是细胞生长调控的核心问题．对SV40病毒和酵母体系的研究为阐明真核生物的复制起始机制及其与细胞周期的关系提供了线索．目前,与DNA复制起始有关的多种蛋白质因子（如核蛋白P₁,DNA单链结合蛋白,DNA聚合酶α,增殖细胞核抗原等）的作用机理逐渐明朗,周期依赖的调控特点得到了证实文章着重介绍了DNA复制起始在细胞周期中的两个调控点及各种周期蛋白在该点的作用,文中还涉及复制起始异常与肿瘤发生的关系.     

组蛋白修饰在染色质复制过程中的作用

真核生物中的DNA复制,不但要保证DNA编码的基因组信息高保真复制,也要保证染色质结构所蕴含的表观遗传组稳定传递,这个过程对于维持基因组的完整性和稳定性至关重要。时至今日,人们对DNA复制的机制已经有了深入的认识,但是对染色质复制以及表观遗传信息传递的了解才刚刚开始。组蛋白是染色质结构中最主要的蛋白组成部分,其上面丰富的转录后修饰是表观遗传调控的核心方式之一。从最近几年组蛋白的修饰研究进展入手,主要综述在DNA复制过程中组蛋白修饰如何参与染色质复制的调控。     

HepA-相关蛋白——一种复制叉结合因子

HepA-相关蛋白(HepA-related protein,HARP),又名SMARCAL1(SWI/SNF-related,matrix-associated,actin-depen-dent regulator of chromatin,subfamily a-like 1),是一种ATP驱动的退火解旋酶,催化解旋的单链DNA重新缠绕成双螺旋。HARP突变可造成一种多系统常染色体隐性疾病——Schimke免疫-骨发育不良(Schimke immuno-osseous dysplasia,SIOD)的发生。HARP可与复制蛋白A(replication protein A,PRA)直接相互作用而被招募到DNA损伤位点以稳定停滞的复制叉,从而保持基因组的完整性。这些研究展示了HARP是DNA损伤反应中的一个关键元件,并且对SIOD发病机制的理解具有重要意义。     

Fanconi anemia proteins stabilize replication forks

Fanconi anemia (FA) is a recessive genetic disorder characterized by hypersensitivity to crosslinking agents that has been attributed to defects in DNA repair and/or replication. FANCD2 and the FA core complex bind to chromatin during DNA replication; however, the role of FA proteins during replication is unknown. Using Xenopus cell-free extracts, we show that FANCL depletion results in defective DNA replication restart following treatment with camptothecin, a drug that results in DSBs during DNA replication. This defect is more pronounced following treatment with mitomycin C, presumably because of an additional role of the FA pathway in DNA crosslink repair. Moreover, we show that chromatin binding of FA core complex proteins during DNA replication follows origin assembly and origin firing and is dependent on the binding of RPA to ssDNA while FANCD2 additionally requires ATR, consistent with FA proteins acting at replication forks. Together, our data suggest that FA proteins play a role in replication restart at collapsed replication forks.     

Dynamics of DNA replication: an ultrastructural study

DNA replication in cells takes place in domains scattered throughout the nucleoplasm. We have characterized the dynamics of DNA synthesis in synchronized mid-S-phase HeLa cells. Saponin-permeabilized cells were allowed to elongate nascent DNA chains in presence of biotin-dUTP for 5, 15, and 30 min (a pulse experiment), or for 5 min followed by an incubation with unlabeled precursors for 10 or 25 min (a pulse-and-chase experiment). The replication foci were then identified in ultrathin sections using immunogold labeling of the incorporated biotin. Total number of particles per nucleus, total scanned area of the nucleus, size, shape, and gold particle number of each labeled cluster, and the density of clusters per nucleus were evaluated. We have demonstrated that as replication proceeds, the labeled sites increase in size up to 240 nm (30 min incorporation) while maintaining a broadly round shape. In pulse-and-chase experiments the labeled DNA was shown to spread to occupy DNA foci of approximately 400 nm in diameter. These results demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view that the synthetic centers are spatially constrained while the chromatin loops are dynamic during DNA synthesis.     

一个新的大肠杆菌单链质粒载体系统

近年来,在重组DNA领域里的一个重要进步,是发展了质粒载体系统。为不同目的建立了不同的载体。噬菌体M13载体系统的建立,大大简化了应用双脱氧末端终止法分析DNA序列和进行特异性点变异的程序。但M13载体仍有局限性,如插入较大的片段常导致载体系统的不稳定,又如操作中常须反复克隆等。许多研究报告指出,将含噬菌体M13或f1复制     

UL52 Primase Interactions in the Herpes Simplex Virus 1 Helicase-Primase Are Affected by Antiviral Compounds and Mutations Causing Drug Resistance

Herpes simplex virus 1 (HSV-1) UL5/8/52 helicase-primase complex is required for DNA unwinding at the replication fork and synthesis of primers during virus replication, and it has become a promising novel target for antiviral therapy. Using molecular cloning, we have identified three separate domains of UL52. Co-immunoprecipitation experiments in extracts from cells transiently expressing HA-tagged UL5, FLAG-UL8, and enhanced GFP-tagged UL52 domains revealed that the N-terminal domain of UL52 primase binds UL5 helicase and the middle domain interacts with the UL8 accessory protein. In addition, an interaction between the single strand DNA-binding protein ICP8 and the UL52 middle domain was observed. The complex between UL5 and UL52 was stabilized by the antiviral compound BAY 54-6322, and mutations providing resistance to the drug obliterate this effect. Our results also suggest a mechanism for accommodating conformational strain resulting from movement of UL5 and UL52 in opposite directions on the lagging strand template, and they identify molecular complexes that can be further examined by structural biology techniques to resolve the mechanism of primer synthesis during herpesvirus replication. Finally, they help to explain the mechanism of action of a novel class of antiviral compounds currently being evaluated in clinical trials.     

MTA2 sensitizes gastric cancer cells to PARP inhibition by induction of DNA replication stress

Poly (ADP-ribose) polymerase (PARP) inhibitor olaparib selectively kills cancer cells with BRCA-deficiency and is approved for BRCA-mutated breast, ovarian and pancreatic cancers by FDA. However, phase III study of olaparib failed to show a significant improvement in overall survival in patients with gastric cancer (GC). To discover an effective biomarker for GC patient-selection in olaparib treatment, we analyzed proteomic profiling of 12 GC cell lines. MTA2 was identified to confer sensitivity to olaparib by aggravating olaparib-induced replication stress in cancer cells. Mechanistically, we applied Cleavage Under Targets and Tagmentation assay to find that MTA2 proteins preferentially bind regions of replication origin-associated DNA sequences, which could be enhanced by olaparib treatment. Furthermore, MTA2 was validated here to render cancer cells susceptible to combination of olaparib with ATR inhibitor AZD6738. In general, our study identified MTA2 as a potential biomarker for olaparib sensitivity by aggravating olaparib-induced replication stress.     

Factors influencing the replication of somatic coliphages in the water environment

The potential replication of somatic coliphages in the environment has been considered a drawback for their use as viral indicators, although the extent to which this affects their numbers in environmental samples has not been assessed. In this study, the replication of somatic coliphages in various conditions was assayed using suspensions containing naturally occurring somatic coliphages and Escherichia coli WG5, which is a host strain recommended for detecting somatic coliphages. The effects on phage replication of exposing strain WG5 and phages to a range of physiological conditions and the effects of the presence of suspended particles or other bacteria were also assayed. Phage replication was further tested using a strain of Klebsiella terrigena and naturally occurring E. coli cells as hosts. Our results indicate that threshold densities of both host bacterium and phages should occur simultaneously to ensure appreciable phage replication. Host cells originating from a culture in the exponential growth phase and incubation at 37 degrees C were the best conditions for phage replication in E. coli WG5. In these conditions the threshold densities required to ensure phage replication were about 10(4) host cells/ml and 10(3) phages/ml, or 10(3) host cells/ml and 10(4) phages/ml, or intermediate values of both. The threshold densities needed for phage replication were higher when the cells proceeded from a culture in the stationary growth phase or when suspended particles or other bacteria were present. Furthermore E. coli WG5 was more efficient in supporting phage replication than either K. terrigenae or E. coli cells naturally occurring in sewage. Our results indicate that the phage and bacterium densities and the bacterial physiological conditions needed for phage replication are rarely expected to be found in the natural water environments.     

G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding

The evolutionarily conserved G-quadruplexes (G4s) are faithfully inherited and serve a variety of cellular functions such as telomere maintenance, gene regulation, DNA replication initiation, and epigenetic regulation. Different from the Watson-Crick base-pairing found in duplex DNA, G4s are formed via Hoogsteen base pairing and are very stable and compact DNA structures. Failure of untangling them in the cell impedes DNA-based transactions and leads to genome instability. Cells have evolved highly specific helicases to resolve G4 structures. We used a recombinant nuclear form of Saccharomyces cerevisiae Pif1 to characterize Pif1-mediated DNA unwinding with a substrate mimicking an ongoing lagging strand synthesis stalled by G4s, which resembles a replication origin and a G4-structured flap in Okazaki fragment maturation. We find that the presence of G4 may greatly stimulate the Pif1 helicase to unwind duplex DNA. Further studies reveal that this stimulation results from G4-enhanced Pif1 dimerization, which is required for duplex DNA unwinding. This finding provides new insights into the properties and functions of G4s. We discuss the observed activation phenomenon in relation to the possible regulatory role of G4s in the rapid rescue of the stalled lagging strand synthesis by helping the replicator recognize and activate the replication origin as well as by quickly removing the G4-structured flap during Okazaki fragment maturation.     

Complete nucleotide sequence of a cryptic plasmid, pbaw301, from the ruminai anaerobe Ruminococcus flavefaciens R13e2

Abstract The complete nucleotide sequence of a cryptic plasmid designated pBAW301, from the Gram-positive ruminai bacterium Ruminococcus flavefaciens R13e2, has been determined. This plasmid is 1768 bp in size and has an overall G+C content of 43.5%. Computer analysis of the sequence data revealed an open reading frame, ORF1 (256 amino acids), which is similar to the Rep protein of the Bacillus borstelensis plasmid pHT926. ORF1 is preceded by Shine-Dalgarno and Escherichia coli —10 and —35 like sequences. Nine smaller open reading frames showed no significant homologies to known protein sequences. Analysis of replication intermediates and the nucleotide sequence indicate that the plasmid does not replicate by a rolling-circle mode of replication similar to other plasmids from Gram-positive bacteria. Moreover, sequences typical of theta replication origins were not found in the nucleotide sequence of pBAW301. These data suggest that this plasmid either replicates by an as yet undescribed mechanism, or represents a new class of theta replicating plasmids.