Biochemical characterization of Periplaneta fuliginosa densovirus non-structural protein NS1

The non-structural (NS) proteins of parvoviruses are involved in essential steps of the viral life cycle. Various biochemical functions, such as ATP binding, ATPase, site-specific DNA binding and nicking, and helicase activities, have been assigned to the protein NS1. Compared with the non-structural proteins of the vertebrate parvoviruses, the NS proteins of the Densovirinae have not been well characterized. Here, we describe the biochemical properties of NS1 of Periplaneta fuliginosa densovirus (PfDNV). We have expressed and purified NS1 using a baculovirus system and analyzed its enzymatic activity. The purified recombinant NS1 protein possesses ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg(2+) or Mn(2+) as a cofactor. The ATPase activity of NS1 can be efficiently stimulated by single-stranded DNA. The ATPase coupled helicase activity was detected on blunt-ended double-stranded oligonucleotide substrate. Using South-Western and Dot-spot assays, we identified a DNA fragment that is recognized specifically by the recombinant NS1 protein. The fragment consists of (CAC)(4) and is located on the hairpin region of the terminal palindrome. The domain for DNA binding was defined to the amino-terminal region (amino acids 1-250). In addition, we found that NS1 can form oligomeric complexes in vivo and in vitro. Mutagenesis analysis showed that ATP binding is necessary for oligomerization. Based on these results, it seems that PfDNV NS1, a multifunctional protein, plays an important role in viral DNA replication comparable to those of vertebrate parvovirus initiator proteins.     

E1 protein of human papillomavirus is a DNA helicase/ATPase.

Replication of human papillomavirus (HPV) DNA requires the viral proteins E1 and E2. Amino acid similarities to SV40 large-T antigen had suggested that E1 is a DNA helicase/ATPase involved in initiating viral DNA replication, and this has recently been shown for bovine papillomavirus type 1 (BPV-1) E1 protein. However, in vitro analysis of HPV E1 has been hampered by the inability to produce purified protein using heterologous expression systems. We have succeeded in demonstrating ATPase and DNA helicase activities in purified HPV E1, expressed in E. coli as a maltose-binding protein fusion (MBP-E1), for the first time. As further confirmation that the ATPase and DNA helicase activities are due to E1 and not contaminating E. coli enzymes, we have shown that a fusion protein containing an amino acid change (E1 Pro-479 to Ser), predicted to inactivate ATP-binding, has impaired activities. We have carried out a structure prediction analysis which suggests that E1 may form two domains: a relatively open N-terminal domain (residues 1-125), and a highly structured C-terminal domain (170-649), with an intermediate region (125-170) predicted to form an inter-domain linker. This is consistent with the proteolytic susceptibility of MBP-E1 at a site 15-20 kD from the N-terminus of E1, and the accumulation of a 58 kD C-terminal fragment of E1. We speculate that the N-terminal domain is involved in DNA-binding, while the C-terminal 58 kD may constitute a distinct enzymatic domain. HPV E1 is of interest as a therapeutic target and the availability of pure enzyme will be invaluable in the search for antiviral compounds.     

Modulation of enzymatic activities of Escherichia coli DnaB helicase by single-stranded DNA-binding proteins

The modulation of enzymatic activities of Escherichia coli DnaB helicase by homologous and heterologous single-stranded DNA-binding proteins (SSBs) and its DNA substrates were analyzed. Although DnaB helicase can unwind a variety of DNA substrates possessing different fork-like structures, the rate of DNA unwinding was significantly diminished with substrates lacking a 3′ fork. A 5 nt fork appeared to be adequate to attain the maximum rate of DNA unwinding. Efficient helicase action of DnaB requires the participation of SSBs. Studies involving heterologous SSBs demonstrated that they can stimulate the helicase activity of DnaB protein under certain conditions. However, this stimulation occurs in a manner distinctly different from that observed with cognate E.coli SSB. The E.coli SSB was found to stimulate the helicase activity over a wide range of SSB concentrations and was unique in its strong inhibition of single-stranded DNA-dependent ATPase activity when uncoupled from the DNA helicase activity. In the presence of a helicase substrate, the ATPase activity of DnaB helicase remained uninhibited. Thus, E.coli SSB appears to coordinate and couple the ATPase activity to the DNA helicase activity by suppressing unproductive ATP hydrolysis by DnaB helicase.     

Characterization of the minimal DNA binding domain of the human papillomavirus e1 helicase: fluorescence anisotropy studies and characterization of a dimerization-defective mutant protein

The E1 helicase of papillomaviruses is required for replication of the viral double-stranded DNA genome, in conjunction with cellular factors. DNA replication is initiated at the viral origin by the assembly of E1 monomers into oligomeric complexes that have unwinding activity. In vivo, this process is catalyzed by the viral E2 protein, which recruits E1 specifically at the origin. For bovine papillomavirus (BPV) E1 a minimal DNA-binding domain (DBD) has been identified N-terminal to the enzymatic domain. In this study, we characterized the DBD of human papillomavirus 11 (HPV11), HPV18, and BPV E1 using a quantitative DNA binding assay based on fluorescence anisotropy. We found that the HPV11 DBD binds DNA with an affinity and sequence requirement comparable to those of the analogous domain of BPV but that the HPV18 DBD has a higher affinity for nonspecific DNA. By comparing the DNA-binding properties of a dimerization-defective protein to those of the wild type, we provide evidence that dimerization of the HPV11 DBD occurs only on two appropriately positioned E1 binding-sites and contributes approximately a 10-fold increase in binding affinity. In contrast, the HPV11 E1 helicase purified as preformed hexamers binds DNA with little sequence specificity, similarly to a dimerization-defective DBD. Finally, we show that the amino acid substitution that prevents dimerization reduces the ability of a longer E1 protein to bind to the origin in vitro and to support transient HPV DNA replication in vivo, but has little effect on its ATPase activity or ability to oligomerize into hexamers. These results are discussed in light of a model of the assembly of replication-competent double hexameric E1 complexes at the origin.     

DNA helicase activity is associated with the replication initiator protein rep of tomato yellow leaf curl geminivirus

The Rep protein of tomato yellow leaf curl Sardinia virus (TYLCSV), a single-stranded DNA virus of plants, is the replication initiator essential for virus replication. TYLCSV Rep has been classified among ATPases associated with various cellular activities (AAA+ ATPases), in superfamily 3 of small DNA and RNA virus replication initiators whose paradigmatic member is simian virus 40 large T antigen. Members of this family are DNA- or RNA-dependent ATPases with helicase activity necessary for viral replication. Another distinctive feature of AAA+ ATPases is their quaternary structure, often composed of hexameric rings. TYLCSV Rep has ATPase activity, but the helicase activity, which is instrumental in further characterization of the mechanism of rolling-circle replication used by geminiviruses, has been a longstanding question. We present results showing that TYLCSV Rep lacking the 121 N-terminal amino acids has helicase activity comparable to that of the other helicases: requirements for a 3' overhang and 3'-to-5' polarity of unwinding, with some distinct features and with a minimal AAA+ ATPase domain. We also show that the helicase activity is dependent on the oligomeric state of the protein.     

The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.     

Evidence for a functional monomeric form of the bacteriophage T4 DdA helicase. Dda does not form stable oligomeric structures

The active form of many helicases is oligomeric, possibly because oligomerization provides multiple DNA binding sites needed for unwinding of DNA. In order to understand the mechanism of the bacteriophage T4 Dda helicase, the potential requirement for oligomerization was investigated. Chemical cross-linking and high pressure gel filtration chromatography provided little evidence for the formation of an oligomeric species. The specific activity for ssDNA stimulated ATPase activity was independent of Dda concentration. Dda was mutated to produce an ATPase-deficient protein (K38A Dda) by altering a residue within a conserved, nucleotide binding loop. The helicase activity of K38A Dda was inactivated, although DNA binding properties were similar to Dda. In the presence of limiting DNA substrate, the rate of unwinding by Dda was not changed; however, the amplitude of product formation was reduced in the presence of increasing concentrations of K38A Dda. The reduction was between that expected for a monomeric or dimeric helicase based on simple competition for substrate binding. When unwinding of DNA was measured in the presence of excess DNA substrate, addition of K38A Dda caused no reduction in the observed rate for strand separation. Taken together, these results indicate that oligomerization of Dda is not required for DNA unwinding.     

Characterization of human papillomavirus type 11 E1 and E2 proteins expressed in insect cells.

The study of human papillomavirus replication has been hampered by the lack of an in vitro system which reliably supports virus replication. Recent results from the bovine papillomavirus (BPV) system indicate that the E1 and E2 proteins are the only viral gene products required for replication. By analogy with simian virus 40 large T antigen, E1 is thought to possess ATPase and helicase activity, which may play a direct role in viral DNA replication. The precise role of E2 is unclear, but it may function in part to help localize E1 to the replication origin. We have initiated a study of replication in the human papillomavirus type 11 system which, by analogy to BPV, has focused on the E1 and E2 proteins of this virus. We have expressed the full-length E1 and E2 proteins in Sf9 insect cells by using a baculovirus expression vector. Both the 80-kDa E1 protein and the 42.5-kDa E2 protein are nuclear phosphoproteins. The E1 and E2 proteins form a heteromeric complex within the insect cells, and both proteins localize to a DNA fragment which contains the viral origin of replication. In addition, we have detected an E1-associated ATPase and GTPase activity, which is likely part of an energy-generating system for the helicase activity which is predicted for this protein. The human papillomavirus type 11 E1 and E2 proteins possess the same replication-associated activities exhibited by the corresponding BPV proteins, suggesting that the replication activities of these viruses are tightly conserved.     

SCFhFBH1 can act as helicase and E3 ubiquitin ligase

In our previous study, we found that a human F-box DNA helicase, named hFBH1, interacted with SKP1 to form an SCF (SKP1–Cul1–F-box protein) complex together with CUL1 and ROC1 in an F-box-dependent manner. The complex immunoprecipitated from crude cell extracts catalyzed polyubiquitin formation in the presence of the ubiquitin-activating and ubiquitin-conjugating enzymes, E1 and E2, respectively. In this report, we characterized the enzymatic properties of the recombinant SCF^hFBH1 complex purified from insect cells expressing hFBH1, SKP1, CUL1 and ROC1. The SCF^hFBH1 complex was isolated as a single tight complex that retained DNA helicase, DNA-dependent ATPase and E3 ubiquitin ligase activities. The helicase and ATPase activities residing in the SCF^hFBH1 complex were indistinguishable from those of the hFBH1 protein alone. Moreover, the ubiquitin ligase activity of the SCF^hFBH1 complex was hardly affected by single-stranded or double-stranded DNA. The multiple activities present in this complex act independently of each other, suggesting that the SCF^hFBH1 complex can catalyze a ubiquitination reaction while acting as a DNA helicase or translocating along DNA. The potential roles of the SCF^hFBH1 complex in DNA metabolism based upon the enzymatic activities associated with this complex are discussed.     

Characterization and mutational analysis of the RecQ core of the bloom syndrome protein

Bloom syndrome protein forms an oligomeric ring structure and belongs to a group of DNA helicases showing extensive homology to the Escherichia coli DNA helicase RecQ, a suppressor of illegitimate recombination. After over-production in E.coli, we have purified the RecQ core of BLM consisting of the DEAH, RecQ-Ct and HRDC domains (amino acid residues 642-1290). The BLM(642-1290) fragment could function as a DNA-stimulated ATPase and as a DNA helicase, displaying the same substrate specificity as the full-size protein. Gel-filtration experiments revealed that BLM(642-1290) exists as a monomer both in solution and in its single-stranded DNA-bound form, even in the presence of Mg(2+) and ATPgammaS. Rates of ATP hydrolysis and DNA unwinding by BLM(642-1290) showed a hyperbolic dependence on ATP concentration, excluding a co-operative interaction between ATP-binding sites. Using a lambda Spi(-) assay, we have found that the BLM(642-1290) fragment is able to partially substitute for the RecQ helicase in suppressing illegitimate recombination in E.coli. A deletion of 182 C-terminal amino acid residues of BLM(642-1290), including the HRDC domain, resulted in helicase and single-stranded DNA-binding defects, whereas kinetic parameters for ATP hydrolysis of this mutant were close to the BLM(642-1290) values. This confirms the prediction that the HRDC domain serves as an auxiliary DNA-binding domain. Mutations at several conserved residues within the RecQ-Ct domain of BLM reduced ATPase and helicase activities severely as well as single-stranded DNA-binding of the enzyme. Together, these data define a minimal helicase domain of BLM and demonstrate its ability to act as a suppressor of illegitimate recombination.     

Cysteine 111 affects coupling of single-stranded DNA binding to ATP hydrolysis in the herpes simplex virus type-1 origin-binding protein

Herpes simplex virus type-1 origin-binding protein (UL9 protein) initiates viral replication by unwinding the origins. It possesses sequence-specific DNA-binding activity, single-stranded DNA-binding activity, DNA helicase activity, and ATPase activity that is strongly stimulated by single-stranded DNA. We have examined the role of cysteines in its action as a DNA helicase. The DNA helicase and DNA-dependent ATPase activities of UL9 protein were stimulated by reducing agent and specifically inactivated by the sulfhydryl-specific reagent N-ethylmaleimide. To identify the cysteine responsible for this phenomenon, a conserved cysteine in the vicinity of the ATP-binding site (cysteine 111) was mutagenized to alanine. UL9C111A protein exhibits defects in its DNA helicase and DNA-dependent ATPase activities and was unable to support origin-specific DNA replication in vivo. A kinetic analysis indicates that these defects are due to the inability of single-stranded DNA to induce high affinity ATP binding in UL9C111A protein. The DNA-dependent ATPase activity of UL9C111A protein is resistant to N-ethylmaleimide, while its DNA helicase activity remains sensitive. Accordingly, sensitivity of UL9 protein to N-ethylmaleimide is due to at least two cysteines. Cysteine 111 is involved in coupling single-stranded DNA binding to ATP-binding and subsequent hydrolysis, while a second cysteine is involved in coupling ATP hydrolysis to DNA unwinding.     

Multimeric BLM is dissociated upon ATP hydrolysis and functions as monomers in resolving DNA structures

Bloom (BLM) syndrome is an autosomal recessive disorder characterized by an increased risk for many types of cancers. Previous studies have shown that BLM protein forms a hexameric ring structure, but its oligomeric form in DNA unwinding is still not well clarified. In this work, we have used dynamic light scattering and various stopped-flow assays to study the active form and kinetic mechanism of BLM in DNA unwinding. It was found that BLM multimers were dissociated upon ATP hydrolysis. Steady-state and single-turnover kinetic studies revealed that BLM helicase always unwound duplex DNA in the monomeric form under conditions of varying enzyme and ATP concentrations as well as 3′-ssDNA tail lengths, with no sign of oligomerization being discerned. Measurements of ATPase activity further indicated that BLM helicase might still function as monomers in resolving highly structured DNAs such as Holliday junctions and D-loops. These results shed new light on the underlying mechanism of BLM-mediated DNA unwinding and on the molecular and functional basis for the phenotype of heterozygous carriers of BLM syndrome.     

Modular architecture of the hexameric human mitochondrial DNA helicase

We have probed the structure of the human mitochondrial DNA helicase, an enzyme that uses the energy of nucleotide hydrolysis to unwind duplex DNA during mitochondrial DNA replication. This novel helicase shares substantial amino acid sequence and functional similarities with the bacteriophage T7 primase-helicase. We show in velocity sedimentation and gel filtration analyses that the mitochondrial DNA helicase exists as a hexamer. Limited proteolysis by trypsin results in the production of several stable fragments, and N-terminal sequencing reveals distinct N and C-terminal polypeptides that represent minimal structural domains. Physical analysis of the proteolytic products defines the region required to maintain oligomeric structure to reside within amino acid residues approximately 405-590. Truncations of the N and C termini affect differentially DNA-dependent ATPase activity, and whereas a C-terminal domain polypeptide is functional, an N-terminal domain polypeptide lacks ATPase activity. Sequence similarity and secondary structural alignments combined with biochemical data suggest that amino acid residue R609 serves as the putative arginine finger that is essential for ATPase activity in ring helicases. The hexameric conformation and modular architecture revealed in our study document that the mitochondrial DNA helicase and bacteriophage T7 primase-helicase share physical features. Our findings place the mitochondrial DNA helicase firmly in the DnaB-like family of replicative DNA helicases.     

Helicase-defective RuvB(D113E) promotes RuvAB-mediated branch migration in vitro.

In Escherichia coli, the RuvA and RuvB proteins interact at Holliday junctions to promote branch migration leading to the formation of heteroduplex DNA. RuvA provides junction-binding specificity and RuvB drives ATP-dependent branch migration. Since RuvB contains sequence motifs characteristic of a DNA helicase and RuvAB exhibit helicase activity in vitro, we have analysed the role of DNA unwinding in relation to branch migration. A mutant RuvB protein, RuvB(D113E), mutated in helicase motif II (the DExx box), has been purified to homogeneity. The mutant protein forms hexameric rings on DNA similar to those formed by wild-type protein and promotes branch migration in the presence of RuvA. However, RuvB(D113E) exhibits reduced ATPase activity and is severely compromised in its DNA helicase activity. Models for RuvAB-mediated branch migration that invoke only limited DNA unwinding activity are proposed.     

Inhibition of DNA helicase II unwinding and ATPase activities by DNA-interacting ligands. Kinetics and specificity.

Although DNA helicases play important roles in the processing of DNA, little is known about the effects of DNA-interacting ligands on these helicases. Therefore, the effects of a wide variety of DNA-binding ligands on the unwinding and ATPase reactions catalyzed by Escherichia coli DNA helicase II were examined. DNA minor groove binders and simple DNA intercalators did not inhibit helicase II. However, DNA intercalators, such as mitoxantrone and nogalamycin, which position functionalities in the major groove upon binding duplex DNA, were potent inhibitors of helicase II. To determine the mechanism by which mitoxantrone inhibited helicase II, the unwinding and DNA-dependent ATPase activities of helicase II were measured using a spectrum of double- and single-stranded DNA substrates. Using either a 71-base pair (bp) M13mp7 partially duplexed DNA substrate or a 245-bp bluntended, fully duplexed DNA substrate, the apparent Ki value for inhibition by mitoxantrone of both the unwinding and ATPase reactions was approximately 1 microM for both substrates, suggesting that the mechanism of inhibition of helicase II by mitoxantrone is the same for both substrates and requires the presence of double-stranded structure. To strengthen this conclusion, the ability of mitoxantrone to inhibit the DNA-dependent ATPase activity of helicase II was determined using two single-stranded substrates, poly(dT) and the 245-bp substrate after heat denaturation. Using either substrate, mitoxantrone inhibited the ATPase activity of helicase II far less effectively. Thus, these results indicate that the intercalation of mitoxantrone into double-stranded DNA, with accompanying placement of functionalities in the major groove, generates a complex that impedes helicase II, resulting in both inhibition of ATP hydrolysis and unwinding activity. Furthermore, we report here that DNA-binding ligands inhibit the unwinding activity of helicases I and IV and Rep protein from E. coli, demonstrating that the inhibition observed for helicase II is not unique to this enzyme.     

Mechanism of DnaB helicase of Escherichia coli: structural domains involved in ATP hydrolysis, DNA binding, and oligomerization.

We describe the delineation of three distinct structural domains of the DnaB helicase of Escherichia coli: domain alpha, amino acid residues (aa) 1-156; domain beta, aa 157-302; and domain gamma, aa 303-471. Using mutants with deletion in these domains, we have examined their role(s) in hexamer formation, DNA-dependent ATPase, and DNA helicase activities. The mutant DnaBbetagamma protein, in which domain alpha was deleted, formed a hexamer; whereas the mutant DnaBalphabeta, in which domain gamma was deleted, could form only dimers. The dimerization of DnaBalphabeta was Mg(2+) dependent. These data suggest that the oligomerization of DnaB helicase involves at least two distinct protein-protein interaction sites; one of these sites is located primarily within domain beta (site 1), while the other interaction site is located within domain gamma (site 2). The mutant DnaBbeta, a polypeptide of 147 aa, where both domains alpha and gamma were deleted, displayed a completely functional ATPase activity. This domain, thus, constitutes the "central catalytic domain" for ATPase activity. The ATPase activity of DnaBalphabeta was kinetically comparable to that of DnaBbeta, indicating that domain alpha had little or no influence on the ATPase activity. In both cases, the ATPase activities were DNA independent. DnaBbetagamma had a DNA-dependent ATPase activity that was kinetically comparable to the ATPase activity of wild-type DnaB protein (wtDnaB), indicating a specific role for C-terminal domain gamma in enhancement of the ATPase activity of domain beta as well as in DNA binding. Mutant DnaBbetagamma, which lacked domain alpha, was devoid of any helicase activity pointing to a significant role for domain alpha. The major findings of this study are (i) domain beta contained a functional ATPase active site; (ii) domain gamma appeared to be the DNA binding domain and a positive regulator of the ATPase activity of domain beta; (iii) although domain alpha did not have any significant effect on the ATPase, DNA binding activities, or hexamer formation, it definitely plays a pivotal role in transducing the energy of ATP hydrolysis to DNA unwinding by the hexamer; and (iv) all three domains are required for helicase activity.     

甲磺酸培氟沙星抑制大肠杆菌RecQ解旋酶的体外DNA结合、解链和ATPase活性

RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族,参与DNA复制、修复、重组、转录及维持端粒稳定等细胞代谢过程,在维持染色体稳定性与完整性中起着重要作用．甲磺酸培氟沙星(pefloxacin mesylate,PFM)是一种新型氟喹诺酮类抗菌药物,对一些革兰氏阴性菌具有明显的杀菌效果,临床上已广泛使用．本研究利用荧光偏振、自由磷检测技术研究PFM对大肠杆菌RecQ解旋酶的DNA结合活性、解链活性、ATPase活性的影响．结果表明,低浓度PFM可促进大肠杆菌RecQ解旋酶与ssDNA、dsDNA结合,达到一定量后PFM则抑制酶与DNA底物的结合,这种影响与DNA底物有关;PFM对RecQ解旋酶的DNA解链活性和ATP酶活性都具有抑制作用,但其抑制的效果有极显著差异(P<0.01)：比较PFM对两种活性抑制的Ci值(对解链活性抑制的Ci值为(1.5±0.2) μmol/L,对ATP酶活性抑制的Ci值为(0.010±0.005) μmol/L)可知,PFM对大肠杆菌RecQ解旋酶ATPase活性的抑制强于其解链活性. 这些结果可为研究以DNA解旋酶为药物靶标的分子机理奠定相关理论基础．     

Hepatitis C virus NS3 helicase forms oligomeric structures that exhibit optimal DNA unwinding activity in vitro

HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNA unwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nm) is equimolar with the substrate, a small burst amplitude of approximately 8 nm is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only approximately 10% of NS3 helicase domain and approximately 20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of full-length NS3 is part of an oligomeric species in vitro.     

大肠杆菌RecQ解旋酶的表达纯化和生物学活性研究

RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族,在维持染色体的稳定性中起着重要的作用.人类RecQ家族解旋酶突变会导致几种与癌症有关的疾病.本研究旨在诱导大肠杆菌RecQ解旋酶体外表达,并应用生物化学和生物物理学技术研究大肠杆菌RecQ解旋酶的生物学活性. 体外诱导表达获得纯度达90% 以上并具有高活性的大肠杆菌重组RecQ解旋酶,其可溶性好;经生物学活性分析显示具有DNA结合活性、ATP依赖的DNA解链活性、DNA依赖的ATP酶活性. 较之双链DNA（dsDNA）,大肠杆菌RecQ解旋酶更容易与单链DNA(ssDNA)结合( P<0.01 ),但与长度不同的dsDNA的结合特性有差异(P<0.01)而与ssDNA没有差异(P>0.05);大肠杆菌RecQ解旋酶对3种dsDNA的解链速率不同(P<0.05);大肠杆菌RecQ解旋酶的ATP酶活性与辅助因子ssDNA长度也呈正相关(P<0.01). 这些研究结果将有助于阐明大肠杆菌RecQ解旋酶的分子作用机制,并为研究RecQ解旋酶家族其它成员的结构与功能提供帮助.     

Replication-associated activities of purified human papillomavirus type 11 E1 helicase

Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.