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1.
对EcoRI作用于DNA的新认识邹国林皮新春(武汉大学生物化学与生物物理学系,武汉430072)关键词蛋白质与核酸相互作用EcoRI模体蛋白质与核酸的相互作用是分子生物学研究的中心问题之一。当前以阐明DNA结合蛋白的结构与蛋白质-DNA复合物的结构为... 相似文献
2.
在植物中发现的锌指蛋白可以分成几种类型[1] 。其中经典的锌指蛋白是TFⅢA型锌指蛋白 ,又称Cys2 /His2 或C2H2 ,模体为CX2— 4CX3 FX5 LX2 HX3— 5 H ,其中的两个Cys和两个His与锌原子形成配位键 ,进而形成包含一个β发夹和一个α螺旋的紧密指状结构 (图 1 ) [2 ] 。曾经认为锌指结构主要介导蛋白质与DNA的互作 ,其中α螺旋与DNA的大沟发生接触 ,然而最近发现有的转录因子中的一些TFⅢA锌指只与蛋白质发生互作 ,而同一转录因子中的其他TFⅢA锌指则与DNA发生互作。第一个在植物中发现的TFⅢA… 相似文献
3.
牛病毒性腹泻病毒基因组cDNA文库的构建 总被引:1,自引:0,他引:1
以牛病毒性腹泻病毒(BVDV)┥nL株的基因组RNA为模板,经逆向转录酶作用,合成第一链cDNA,再以RNadse/H与DNA聚合酶I联合作用合成dscDNA,并以dC同聚物尾化。pUC8DNA在Pst I酶解后,以dG同聚物尾化,两者退火构成重组质粒,转化到E.coli JM101受体菌中,另以γ-^32P-ATP标记BVDV RNA制备探针,通过菌落原位杂交筛选重组子。酶切分配表明重组质粒插入 相似文献
4.
豌豆卷须肌动蛋白Ⅱ类异型体cDNA克隆的序列分析 总被引:4,自引:0,他引:4
分析15个豌豆卷须肌动蛋白cDNA 克隆的限制性内切酶图谱,发现在豌豆卷须中至少存在三类肌动蛋白异型体,分别命名为Ⅰ类(PEAc Ⅰ)、Ⅱ类(PEAc Ⅱ)和Ⅲ类(PEAc Ⅲ)异型体.三类异型体的克隆数目分别为10、4和1个,表明三类异型体在豌豆卷须中的表达是不同的,很可能具有组织或发育阶段的特异性.对Ⅱ类异型体的三个cDNA 克隆PEAc3、PEAc9和PEAc11进行了全序列测定,所测定的序列已被GenBank 数据库所接受.测序结果表明,PEAc3、PEAc9和PEAc11的序列长度分别为1550、1680和1091个核苷酸,其中编码区长1134个核苷酸,编码的氨基酸长度为377(PEAc11缺少编码氨基端前96个氨基酸的核苷酸序列).三个克隆的核苷酸序列完全相同,差别仅在于3′非翻译区的长度不同,即poly(A)的加入位点不同.这说明它们可能是由同一基因转录而来,但转录后的加工过程不同.豌豆卷须中肌动蛋白基因poly(A)加入位点的使用,可能与组织或发育的特异性表达有关.此外,豌豆卷须肌动蛋白三类异型体之间的核苷酸序列同源性为80% ,氨基酸序列同源性为94% . 相似文献
5.
板齿鼠线粒体DNA的研究 总被引:4,自引:0,他引:4
本文应用ApaI、BamHI、BclI、BglI、ClaI、EcoRI、EcoRV、HindII、PstI、PvuII、SacI、ScaI和XbaI等13种限制性内切酶对板齿鼠线粒体DNA(mtDNA)进行限制性片段长度多态(RFLP)分析,并用双酶解法构建其限制性内切酶图谱。结果表明板齿鼠存在3种mtDNA单倍型,可通过限制酶PvuII、HindII和ApaI区分,呈现DNA多态性和种内遗传变异。与小家鼠、褐家鼠mtDNA限制性片段的数据相比较,板齿鼠和这两种鼠mtDNA存在明显差异。板齿鼠mtDNA限制性内切酶图谱的建立,为进一步系统研究鼠科动物的遗传分化提供了依据。 相似文献
6.
大鼠α-酰胺酶在变铅青链霉菌中的克隆及表达研究 总被引:1,自引:0,他引:1
α-酰胺酶(α-amidase,α-AE)催化神经和内分泌系统中活性多肽的C-端酰胺化,多多肽的生物活性至关重要。以大鼠心房组织的总RNA为模板,采用RT-PCR技术扩增获得编码α-酰胺酶的cDNA,并进行了克隆和测序。为了使α-酰胺酶能在链霉菌中分泌表达,将其cDNA与链霉菌酷氮酶酶(melC1)信号的编码序列融合得到融合mel/AE,将mel/AE插入链霉菌质粒pIJ680,获得重组质粒pIJ 相似文献
7.
从印度木薯花叶病毒(ICMV)侵染的植物中纯化特异的核酸,经RNAase,DNAasc,Nucle-aseSl,ExonucleaseⅢ和EcoRI酶切,Southern和Dotblots杂交证实,在感病的植株中,存在两种形式的病毒核酸:环状双链DNA和环状单链DNA,后者可能是病毒DNA的(-)链,环状双链DNA经限制性内切酶作用可得2.7kb的线性双链DNA纯化的病毒核酸含DNA1和DNA2两个分子量相近的组份。 相似文献
8.
磁场对质粒pBR322DNA的影响 总被引:6,自引:1,他引:5
讨论了经500mT、900mT、1200mT、1500mT恒磁场作用后的质粒pBR322DNA与Ⅱ型限制性内切酶EcoRⅠ、HindⅢ、BamHⅠSalⅠ、PstⅠ、PuvⅡ、TaqⅠ、BglⅠ、RsaⅠ、HinfⅠ、APaⅠ、KpnⅠ等的单酶解、双酶解及多酶解反应。从琼脂糖电泳分离酶解片段的结果,发现经磁场作用过的pBR322DNA在3361—631bp的区段内产生了一个被HinfⅠ识别的新序列GANTC,证明了磁场作用能引起DNA点突变。 相似文献
9.
栖热菌属热稳定DNA聚合酶 总被引:7,自引:2,他引:5
DNA聚合酶是能够以DNA或RNA为模板 ,在寡核苷酸或蛋白质引物的引导下催化合成DNA的一类酶 ,已经分离纯化的有上百种 ,其中有半数已经被克隆和测序。它们之间在DNA序列、氨基酸序列、二、三级结构方面有较高的同源性 ,且与RNA聚合酶和反转录酶之间也有不同程度的同源性[1~ 4] 。1 DNA聚合酶的分类DNA聚合酶有许多不同的类群 ,如很小的 (3 9kDa) ,来自哺乳动物的修复性单亚基DNA聚合酶 ,巨大的 (可高达 90 0kDa)、多亚基(可达 2 0多个亚基 )的复制性DNA聚合酶 (如Eco聚合酶Ⅲ )。有些DNA聚合酶则… 相似文献
10.
大鼠催乳素基因真核细胞可表达性质粒的构建及应用研究 总被引:4,自引:0,他引:4
735bp的PRLcDNA片段从质粒PRL-SP65#1中回收后,用粘性末端连接法将其重组到真核表达载体pcDNA3上,筛选出正向连接重组体pcDNA3-PRLS和反向连接重组体pcDNA3-PRLAS。将重组体pcDNA3-PRLs和空载体pcDNA3分别转入NIH3T3细胞系,用G418筛选出阳性细胞后与未转染的NIH3T3细胞在加E2和不加E2的情况下,用原位杂交的方法,分别用PRLcDNA探针和原癌基因c-H-rascDNA探针进行检测,未转染的NIH3T3细胞在加E2和不加E2时都几乎无催乳素基因的表达,同样,转入空载体的NIH3T3细胞也无PRL的表达,而转入重组体pcDNA3-PRLS的NIH3T3细胞则有大量的PRL基因的表达,与对照组相比有显著差异(P<0.01)。正常和转入空载体的NIH3T3细胞有一定程度的原癌基因c-H-ras的表达,当分别加入E2和转入重组体pcDNA3-PRLS后,NIH3T3细胞中的c-H-ras基因表达水平都显著升高(P<0.05)。 相似文献
11.
Incorporation of fluorine into proteins has long served as a means of probing structure and function, yet there are few studies that examine the impact of fluorine substitution, particularly at locations distant from the active sites of enzymes. The flexibility of isomeric fluorine incorporation at Phe is used to explore subtle substitution effects on enzyme activity and conformation. The unnatural amino acids o-, m-, and p-fluorophenylalanines were incorporated biosynthetically into the representative PvuII restriction endonuclease. Interestingly, m-fluoro-Phe-PvuII endonuclease exhibits very similar conformational stability to that of the native enzyme, but it exhibits a reproducible, 2-fold higher average specific activity. Given the level of incorporation and the distribution of species, the species of modified enzyme responsible for this increase in specific activity is most likely even faster. Further, moving the fluorine atom from the meta- to the para-position of Phe results in a 4-fold decrease in specific activity and a decrease in conformational stability of 1.5 kcal/mol. Since none of the Phe residues in PvuII endonuclease lies near the DNA recognition or catalytic sites, this differential behavior alludes to the impact of subtle changes in enzyme conformation on endonuclease activity and suggests novel ways to influence catalytic behavior. 相似文献
12.
The first X-ray structures of an intein-DNA complex, that of the two-domain homing endonuclease PI-SceI bound to its 36-base pair DNA substrate, have been determined in the presence and absence of Ca(2+). The DNA shows an asymmetric bending pattern, with a major 50 degree bend in the endonuclease domain and a minor 22 degree bend in the splicing domain region. Distortions of the DNA bound to the endonuclease domain cause the insertion of the two cleavage sites in the catalytic center. DNA binding induces changes in the protein conformation. The two overlapping non-identical active sites in the endonucleolytic center contain two Ca(+2) ions that coordinate to the catalytic Asp residues. Structure analysis indicates that the top strand may be cleaved first. 相似文献
13.
14.
Phage-encoded resolvase T7 endonuclease I is a structure-specific endonuclease. The enzyme acts on a broad spectrum of substrates with a variety of DNA structures. The enzyme is a dimer with two separated catalytic domains connected by an elongated beta-sheet bridge. The activities of enzymes with mutations in the beta-bridge segment were studied. Mutations that did not affect catalytic domain folding and function but did alter the relative positions of these domains retained catalytic activity but with altered specificity and metal ion dependence. Our results suggest that the enzyme recognizes its substrates by DNA conformation exclusion and offer a simple explanation for the broad substrate specificity of phage resolvase. 相似文献
15.
We have determined the structure of PvuII endonuclease complexed with cognate DNA by X-ray crystallography. The DNA substrate is bound with a single homodimeric protein, each subunit of which reveals three structural regions. The catalytic region strongly resembles structures of other restriction endonucleases, even though these regions have dissimilar primary sequences. Comparison of the active site with those of EcoRV and EcoRI endonucleases reveals a conserved triplet sequence close to the reactive phosphodiester group and a conserved acidic pair that may represent the ligands for the catalytic cofactor Mg2+. The DNA duplex is not significantly bent and maintains a B-DNA-like conformation. The subunit interface region of the homodimeric protein consists of a pseudo-three-helix bundle. Direct contacts between the protein and the base pairs of the PvuII recognition site occur exclusively in the major groove through two antiparallel beta strands from the sequence recognition region of the protein. Water-mediated contacts are made in the minor grooves to central bases of the site. If restriction enzymes do share a common ancestor, as has been proposed, their catalytic regions have been very strongly conserved, while their subunit interfaces and DNA sequence recognition regions have undergone remarkable structural variation. 相似文献
16.
Chemical evidence that chromatin DNA exists as 160 base pair beads interspersed with 40 base pair bridges.
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Digestion of rat liver nuclei by an endogenous endonuclease generates double-stranded DNA fragments which are initially about 205 base pairs long, as reported previously by Hewish and Burgoyne. As digestion proceeds, the average size of these fragments is reduced to about 160 base pairs. Electrophoresis under denaturing conditions shows that these DNA fragments contain single strand nicks at ten base intervals. Fifteen bands, 10-150 bases, are clearly resolvable. DNA Fragments of 160 to 200 nucleotides are not resolved as distinct species. The results suggest that the chromosomal subunit contains both a 160 base-pair DNA segment, in a conformation susceptible to single strand nicking at ten base intervals, and a forty base-pair DNA segment in a conformation more uniformly susceptible to endogenous endonuclease activity. This chemical evidence agrees with morphological observations suggesting that chromatin has a "bead and bridge" structure. 相似文献
17.
NAE:I is transformed from DNA endonuclease to DNA topoisomerase and recombinase by a single amino acid substitution. The crystal structure of NAE:I was solved at 2.3 A resolution and shows that NAE:I is a dimeric molecule with two domains per monomer. Each domain contains one potential DNA recognition motif corresponding to either endonuclease or topoisomerase activity. The N-terminal domain core folds like the other type II restriction endonucleases as well as lambda-exonuclease and the DNA repair enzymes MutH and Vsr, implying a common evolutionary origin and catalytic mechanism. The C-terminal domain contains a catabolite activator protein (CAP) motif present in many DNA-binding proteins, including the type IA and type II topoisomerases. Thus, the NAE:I structure implies that DNA processing enzymes evolved from a few common ancestors. NAE:I may be an evolutionary bridge between endonuclease and DNA processing enzymes. 相似文献
18.
Ichiyanagi K Ishino Y Ariyoshi M Komori K Morikawa K 《Journal of molecular biology》2000,300(4):889-901
Inteins possess two different enzymatic activities, self-catalyzed protein splicing and site-specific DNA cleavage. These endonucleases, which are classified as part of the homing endonuclease family, initiate the mobility of their genetic elements into homologous alleles. They recognize long asymmetric nucleotide sequences and cleave both DNA strands in a monomer form. We present here the 2.1 A crystal structure of the archaeal PI-PfuI intein from Pyroccocus furiosus. The structure reveals a unique domain, designated here as the Stirrup domain, which is inserted between the Hint domain and an endonuclease domain. The horseshoe-shaped Hint domain contains a catalytic center for protein splicing, which involves both N and C-terminal residues. The endonuclease domain, which is inserted into the Hint domain, consists of two copies of substructure related by an internal pseudo 2-fold axis. In contrast with the I-CreI homing endonuclease, PI-PfuI possibly has two asymmetric catalytic sites at the center of a putative DNA-binding cleft formed by a pair of four-stranded beta-sheets. DNase I footprinting experiments showed that PI-PfuI covers more than 30 bp of the substrate asymmetrically across the cleavage site. A docking model of the DNA-enzyme complex suggests that the endonuclease domain covers the 20 bp DNA duplex encompassing the cleavage site, whereas the Stirrup domain could make an additional contact with another upstream 10 bp region. For the double-strand break, the two strands in the DNA duplex were cleaved by PI-PfuI with different efficiencies. We suggest that the cleavage of each strand is catalyzed by each of the two non-equivalent active sites. 相似文献
19.
The DNA sequence of the bacteriophage T4 denV gene which encodes the DNA repair enzyme endonuclease V was previously constructed behind the hybrid lambda promoter OLPR in a plasmid vector. The OLPR-denV sequence was subcloned in M13mp18 and used as template to construct site-specific mutations in the denV structural gene in order to investigate structure/function relationships between the primary structure of the protein and its various DNA binding and catalytic activities. The Lys-130 residue of the wild-type endonuclease V has been postulated to be associated with its apurinic endonuclease (AP-endonuclease) activity. The codon for Lys-130 was changed to His-130 or Gly-130, and each denV sequence was subcloned into a pEMBL expression vector. These plasmids were transformed into repair-deficient Escherichia coli (uvrA recA), and the following parameters were examined for cells or cell extracts: expression and accumulation of endonuclease V protein (K-130, H-130, or G-130); survival after UV irradiation; dimer-specific DNA binding; and kinetics of phosphodiester bond scission at pyrimidine dimer sites, dimer-specific N-glycosylase activity, and AP-endonuclease activity. The enzyme's intracellular accumulation was significantly decreased for G-130 and slightly decreased for H-130 despite normal levels of denV-specific mRNA for each mutant. On a molar basis, the endonuclease V gene products generally gave parallel levels of each of the catalytic and binding functions with K-130 greater than H-130 greater than G-130 much greater than control denV-.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
Yamamoto T Iino H Kim K Kuramitsu S Fukui K 《The Journal of biological chemistry》2011,286(49):42337-42348
DNA mismatch repair (MMR) greatly contributes to genome integrity via the correction of mismatched bases that are mainly generated by replication errors. Postreplicative MMR excises a relatively long tract of error-containing single-stranded DNA. MutL is a widely conserved nicking endonuclease that directs the excision reaction to the error-containing strand of the duplex by specifically nicking the daughter strand. Because MutL apparently exhibits nonspecific nicking endonuclease activity in vitro, the regulatory mechanism of MutL has been argued. Recent studies suggest ATP-dependent conformational and functional changes of MutL, indicating that the regulatory mechanism involves the ATP binding and hydrolysis cycle. In this study, we investigated the effect of ATP binding on the structure of MutL. First, a cross-linking experiment confirmed that the N-terminal ATPase domain physically interacts with the C-terminal endonuclease domain. Next, hydrogen/deuterium exchange mass spectrometry clarified that the binding of ATP to the N-terminal domain induces local structural changes at the catalytic sites of MutL C-terminal domain. Finally, on the basis of the results of the hydrogen/deuterium exchange experiment, we successfully identified novel regions essential for the endonuclease activity of MutL. The results clearly show that ATP modulates the nicking endonuclease activity of MutL via structural rearrangements of the catalytic site. In addition, several Lynch syndrome-related mutations in human MutL homolog are located in the position corresponding to the newly identified catalytic region. Our data contribute toward understanding the relationship between mutations in MutL homolog and human disease. 相似文献