大肠杆菌RecQ解旋酶的生物学活性分析

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(P0.01),但与长度不同的dsDNA的结合特性有差异(P0.01)而与ssDNA没有差异(P0.05);大肠杆菌RecQ解旋酶对3种dsDNA的解链速率不同(P0.05);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(P0.01).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。     

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.     

洛美沙星对Bloom综合征解旋酶生物学特性影响的机理研究

Bloom综合征解旋酶(BLM)是RecQ家族DNA解旋酶中的一个重要成员,参与了DNA复制、修复、转录、重组以及端粒的维持等细胞代谢过程,在维持染色体的稳定性中具有重要作用.BLM解旋酶的突变可导致Bloom综合征.Bloom综合征是一种罕见隐性常染色体遗传疾病,患者遗传不稳定,并易患多种类型癌症.洛美沙星(LMX)可以抑制细胞内多种酶,并通过结合DNA干扰DNA代谢,从而治疗多种疾病,但是其具体的作用机理还未完全清楚.运用荧光偏振技术和自由磷检测技术,研究了LMX对BLM642～1290解旋酶DNA结合活性、解链活性和ATP酶活性的影响.运用荧光及紫外吸收光谱法研究了LMX与解旋酶结合的结合常数、结合位点数、作用力类型、结合距离等参数.结果表明,LMX与解旋酶之间能自发进行反应,两种分子有一个结合位点,通过静电引力和疏水作用力形成稳定的BLM-LMX复合物,且解旋酶的内源荧光被LMX静态猝灭,主要原因是非辐射能量转移.在这一过程中,LMX能抑制解旋酶的解链活性和ATP酶活性,而促进解旋酶的DNA结合活性.LMX对BLM解旋酶生物学活性影响的机理可能是LMX使解旋酶通过别构机制影响其ATP酶活性,并使酶的构象维持在较低解链活性的状态,通过抑制ATP催化水解与解链过程的偶联和阻止解旋酶的易位,从而抑制其解链.LMX能够促进解旋酶的DNA结合活性,可能是因为其C-6和C-7上的取代功能基团可以增加酶活力,以及增强药物、酶和DNA的结合,从而形成药物-酶-DNA复合物.这些结果为研究以DNA解旋酶为药物靶标的分子机理和理解喹诺酮类药物的作用机理奠定相关理论基础.     

荧光偏振技术研究Bloom解旋酶催化核心与双链DNA的相互作用

Bloom 综合症(BLM)解旋酶是RecQ家族DNA解旋酶中的一个重要成员,参与了DNA复制、修复、转录、重组以及端粒的维持等细胞代谢过程,在维持染色体的稳定性中具有重要的作用．BLM解旋酶的突变可导致Bloom综合症,患者遗传不稳定易患多种类型癌症．本研究运用荧光偏振技术研究BLM解旋酶催化核心(BLM642-1290)与双链DNA(dsDNA)的相互作用,分析其相关特征参数,了解BLM642-1290解旋酶与dsDNA的结合和解链特性．结果表明,BLM642-1290解旋酶与dsDNA的结合和解链和dsDNA3’末端的单链DNA(ssDNA)长度有关;解旋酶优先结合于dsDNA底物的ssDNA末端,且每分子解旋酶可结合9.6 nt的ssDNA;dsDNA3’末端ssDNA的长度为9.6 nt时,解旋酶的解链效率达到最大且不再随其长度而变化．另外,BLM642-1290解旋酶也能够结合和解链钝末端dsDNA,但其结合亲和力和解链效率低于有3’末端ssDNA的dsDNA．推测BLM642-1290解旋酶在与dsDNA底物结合和解链时是单体形式,可能以尺蠖的形式解开dsDNA．这些结果可为进一步研究BLM解旋酶的功能特征提供理论基础．     

荧光偏振技术研究Bloom解旋酶催化核心与双链DNA的相互作用

Bloom 综合症(BLM)解旋酶是RecQ家族DNA解旋酶中的一个重要成员,参与了DNA复制、修复、转录、重组以及端粒的维持等细胞代谢过程,在维持染色体的稳定性中具有重要的作用．BLM解旋酶的突变可导致Bloom综合症,患者遗传不稳定易患多种类型癌症．本研究运用荧光偏振技术研究BLM解旋酶催化核心(BLM^642～1290)与双链DNA(dsDNA)的相互作用,分析其相关特征参数,了解BLM^642～1290解旋酶与dsDNA的结合和解链特性．结果表明：BLM^642～1290解旋酶与dsDNA的结合和解链与dsDNA 3′端的单链DNA(ssDNA)长度有关;解旋酶优先结合于dsDNA底物的ssDNA末端,且每分子解旋酶可结合9.6 nt的ssDNA;dsDNA 3′端ssDNA的长度为9.6 nt时,解旋酶的解链效率达到最大且不再随其长度而变化．另外,BLM^642～1290解旋酶也能够结合和解链钝末端dsDNA,但其结合亲和力和解链效率低于有3′端ssDNA的dsDNA．推测BLM^642～1290解旋酶在与dsDNA底物结合和解链时是单体形式,可能以尺蠖的形式解开dsDNA．这些结果可为进一步研究BLM解旋酶的功能特征提供理论基础．     

甘草次酸对Bloom解旋酶生物学特性的影响

甘草次酸(glycyrrhetinic acid,GA)是甘草主要活性组分,可诱导肿瘤细胞凋亡,抑制肿瘤细胞生长.然而,其对BLM解旋酶的抑制作用尚未见报道.本文注视甘草次酸对BLM解旋酶构象、二级结构和生化活性的影响.圆二色光谱和紫外光谱分析显示,GA可破坏BLM642-1290解旋酶α-螺旋结构,改变其构象,并具有2个结合位点.采用荧光偏振技术和自由磷检测证明,GA以浓度依赖的方式抑制BLM642-1290解旋酶与底物dsDNA及ssDNA的结合,抑制BLM642-1290解旋酶活性及ATP酶活性,且抑制类型为混合抑制.综上所述,本文证明GA可通过结合BLM解旋酶,改变BLM解旋酶构象,抑制BLM解旋酶与DNA的结合,从而抑制BLM解旋酶的生化活性.我们的发现将对深入认识GA的抗肿瘤作用有新的启示.     

极端嗜热古菌——芝田硫化叶菌DNA解旋酶的分离纯化和性质研究

采用Qsepharose离子交换层析、磷酸纤维素P1 1吸附层析、肝素琼脂糖吸附层析、Su perdex 2 0 0凝胶过滤和PhenylSuperose疏水层析等步骤 ,从嗜酸热芝田硫化叶菌细胞裂解液中分离纯化了一个DNA解旋酶。该解旋酶具有受DNA激活的ATP酶活性。根据SDS PAGE测定结果 ,该酶的分子质量约为 63kD。芝田硫化叶菌DNA解旋酶可以解开底物上 70bp的双链区 ,其解旋活性依赖于双链区旁的单链分叉。该解旋酶的活性依赖于Mg2 + 和ATP的水解 ,在NaCl浓度超过 2 0 0mmol L时受到抑制。该酶的最适pH为 6 7。该酶在 40℃～ 80℃之间均有活性 ,70℃时活性最高。芝田硫化叶菌DNA解旋酶是从古菌中分离得到的第一个天然DNA解旋酶。     

SARS冠状病毒主蛋白酶抑制剂的筛选及抑制动力学研究

对SARS冠状病毒主蛋白酶(SARS-CoV M^pro)进行异源重组表达与提纯,并以其为靶点,利用基于荧光共振能量转移(FRET)技术的体外药物筛选模型,对蛋白酶抑制剂聚焦库96种化合物进行了体外抑制活性的评价,并从动力学的角度探讨筛选出的阳性化合物对SARS-CoV M^pro的抑制能力与机制。结果表明:通过筛选获得抑制率>80%、淬灭率<20%的化合物5种,为P-1-08、P-1-19、P-2-24、P-2-28、P-2-54,其半数有效抑制浓度(IC₅₀)分别为:0.69±0.05μmol/L、1.19±0.41μmol/L、0.14±0.01μmol/L、1.36±0.07μmol/L、0.36±0.03μmol/L。其中化合物P-1-08、P-1-19、P-2-24、P-2-54对SARS冠状病毒主蛋白酶的抑制作用为不可逆抑制,化合物P-2-28的抑制作用为可逆抑制。根据Lineweaver-Burk图和Dixon图的研究,发现化合物P-2-28对SARS冠状病毒主蛋白酶呈竞争性抑制,抑制常数Ki为0.81μmol/L。通过对底物浓度,IC₅₀值及Ki值关系的研究,进一步验证了P-2-28的抑制作用为竞争性抑制。该抑制剂的发现为SARS冠状病毒主蛋白酶抑制剂的研究打下基础,为抗SARS病毒药物开发提供了先导化合物。     

嗜热脱铁去硫弧菌Pif1解旋酶生物学活性的分子特征分析

已知Pif1解旋酶在维持基因组稳定性方面发挥重要作用,且不同生物Pif1解旋酶具有不同的生物学活性;然而迄今为止,嗜热细菌Pif1解旋酶的生物学活性分子特征的研究尚未见报道。本文运用生物化学与生物物理学前沿技术,系统地研究了嗜热脱铁去硫弧菌Pif1解旋酶（Defe.Pif1）结合活性与解旋活性的分子特征。通过原核表达纯化系统,本研究获得纯度95%以上、无标签的Defe.Pif1蛋白。利用荧光偏振技术研究Defe.Pif1结合反应的底物特异性,揭示出Defe.Pif1优先结合ssDNA与G4 DNA,并对含3′-尾链的部分双链底物有较强亲和力,其结合反应底物特异性为：ssDNA > G4 DNA > 3′-ssDNA-dsDNA≈Y-structure > Other substrates。通过快速停留检测技术研究Defe.Pif1的解旋活性,明确其最适解旋温度为50℃,最佳反应溶液为10 mmol/L NaCl、3 mmol/L DTT、3 mmol/L MgCl2及1 mmol/L ATP;进一步的解旋动力学特征分析结果显示,Defe.Pif1可以高效解旋含G4结构的DNA底物,其解旋5′-G4 dsDNA底物时的解旋幅度超过90%,解旋速率也与其解旋5′-ss-dsDNA底物的速率相近,提示Defe.Pif1解旋G4 DNA更接近Bs.Pif1的单体反应模式。此外,本研究还发现Defe.Pif1解旋不同类型复制叉/复制泡底物时拥有独特的解旋倾向性：与解旋其它复制中间体DNA的低效性不同,Defe.Pif1解旋12nt bubble底物的速率与幅度均较高,暗示12nt bubble结构很可能是该解旋酶复制中间体的天然底物。上述结果证明,Defe.Pif1不仅具有Pif1解旋酶家族成员共同的结合与解旋G4 DNA等活性特征;而且作为嗜热细菌的解旋酶,它还具有独特的反应条件与解旋底物特异性。本研究为研究Pif1解旋酶家族其它成员的分子特征与生物功能提供了潜在的研究策略,为阐明此类Pif1解旋酶的分子作用机制奠定实验基础。     

依托泊苷影响HEK293细胞同源重组修复体系

抗肿瘤药物疗效的研究多集中在肿瘤细胞,目前针对正常细胞的研究颇少,有必要建立能进行定量分析的同源重组定量修复体系。我们已建立的模型可以探讨肿瘤药物化疗后对HEK293细胞DSBs修复的继发性后果。通过构建含有带I-SceⅠ酶切位点的同源介导的重组修复底物（homologous direct recombination, HDR）,或单链退火修复（single strand annealing, SSA）底物的细胞株,定量检测依托泊苷 (etoposide,VP-l6)对同源性重组修复(homologous recombination, HR)通路的影响。成功构建了可用于定量检测DNA双链断裂(double-strand break, DSBs)诱导的SSA和HDR修复的正常人HEK293细胞应用模型。细胞毒结果证实,与SSA/293对照组对比,VP-16给药组 16 μmol/L（0.475±0.029 vs 1.000±0.000, P<0.001)细胞活力明显降低;与HDR/293对照组相比,VP-16 给药组16 μmol/L（0.458±0.188 vs 1.000±0.000, P<0.05)细胞活力降低。此外,本研究证实,VP-16抑制SSA修复,VP-16给药组 2 μmol/L与SSA/293对照组相比（0.575%±0.177% vs 1.352%±0.195%, P<0.05）,修复效率降低;VP-16抑制HDR修复,VP-16给药组1 μmol/L与HDR/293对照组修复效率相比（0.305%±0.078% vs. 0.635%± 0.049%,P<0.05),修复效率降低。VP-16诱导DNA损伤的同时,抑制HDR修复和SSA修复,修复效率呈现剂量依赖性。本研究结果可为抗肿瘤药物的临床应用提供某些指导。     

Biochemical characterization of the DNA helicase activity of the escherichia coli RecQ helicase

We demonstrate that RecQ helicase from Escherichia coli is a catalytic helicase whose activity depends on the concentration of ATP, free magnesium ion, and single-stranded DNA-binding (SSB) protein. Helicase activity is cooperative in ATP concentration, with an apparent S(0.5) value for ATP of 200 microm and a Hill coefficient of 3.3 +/- 0.3. Therefore, RecQ helicase utilizes multiple, interacting ATP-binding sites to mediate double-stranded DNA (dsDNA) unwinding, implicating a multimer of at least three subunits as the active unwinding species. Unwinding activity is independent of dsDNA ends, indicating that RecQ helicase can unwind from both internal regions and ends of dsDNA. The K(M) for dsDNA is 0.5-0.9 microm base pairs; the k(cat) for DNA unwinding is 2.3-2.7 base pairs/s/monomer of RecQ helicase; and unexpectedly, helicase activity is optimal at a free magnesium ion concentration of 0.05 mm. Omitting Escherichia coli SSB protein lowers the rate and extent of dsDNA unwinding, suggesting that RecQ helicase associates with the single-stranded DNA (ssDNA) product. In agreement, the ssDNA-dependent ATPase activity is reduced in proportion to the SSB protein concentration; in its absence, ATPase activity saturates at six nucleotides/RecQ helicase monomer and yields a k(cat) of 24 s(-1). Thus, we conclude that SSB protein stimulates RecQ helicase-mediated unwinding by both trapping the separated ssDNA strands after unwinding and preventing the formation of non-productive enzyme-ssDNA complexes.     

Coupling DNA-binding and ATP hydrolysis in Escherichia coli RecQ: role of a highly conserved aromatic-rich sequence

RecQ enzymes are broadly conserved Superfamily-2 (SF-2) DNA helicases that play critical roles in DNA metabolism. RecQ proteins use the energy of ATP hydrolysis to drive DNA unwinding; however, the mechanisms by which RecQ links ATPase activity to DNA-binding/unwinding are unknown. In many Superfamily-1 (SF-1) DNA helicases, helicase sequence motif III links these activities by binding both single-stranded (ss) DNA and ATP. However, the ssDNA-binding aromatic-rich element in motif III present in these enzymes is missing from SF-2 helicases, raising the question of how these enzymes link ATP hydrolysis to DNA-binding/unwinding. We show that Escherichia coli RecQ contains a conserved aromatic-rich loop in its helicase domain between motifs II and III. Although placement of the RecQ aromatic-rich loop is topologically distinct relative to the SF-1 enzymes, both loops map to similar tertiary structural positions. We examined the functions of the E.coli RecQ aromatic-rich loop using RecQ variants with single amino acid substitutions within the segment. Our results indicate that the aromatic-rich loop in RecQ is critical for coupling ATPase and DNA-binding/unwinding activities. Our studies also suggest that RecQ's aromatic-rich loop might couple ATP hydrolysis to DNA-binding in a mechanistically distinct manner from SF-1 helicases.     

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.     

The Escherichia coli RecQ helicase functions as a monomer

The RecQ helicases belong to an important family of highly conserved DNA helicases that play a key role in chromosomal maintenance, and their defects have been shown to lead to several disorders and cancer in humans. In this work, the conformational and functional properties of the Escherichia coli RecQ helicase have been determined using a wide array of biochemical and biophysical techniques. The results obtained clearly indicate that E. coli RecQ helicase is monomeric in solution up to a concentration of 20 microM and in a temperature range between 4 and 37 degrees C. Furthermore, these properties are not affected by the presence of ATP, which is strictly required for the unwinding and translocating activity of the protein, or by its nonhydrolyzable analogue 5'-adenylyl-beta,gamma-imidodiphosphate. Consistent with the structural properties, functional analysis shows that both DNA unwinding activity and single-stranded DNA-stimulated ATPase specific activity were independent of RecQ concentration. The monomeric state was further confirmed by the ATPase-deficient mutants of RecQ protein. The rate of unwinding was unchanged when the wild type RecQ helicase was mixed with the ATPase-deficient mutants, indicating that nonprotein-protein interactions were involved in the unwinding processes. Taken together, these results indicate that RecQ helicase functions as a monomer and provide new data on the structural and functional properties of RecQ helicase that may help elucidate its mechanism action.     

RecQ helicase stimulates both DNA catenation and changes in DNA topology by topoisomerase III

Together, RecQ helicase and topoisomerase III (Topo III) of Escherichia coli comprise a potent DNA strand passage activity that can catenate covalently closed DNA (Harmon, F. G., DiGate, R. J., and Kowalczykowski, S. C. (1999) Mol. Cell 3, 611-620). Here we directly assessed the structure of the catenated DNA species formed by RecQ helicase and Topo III using atomic force microscopy. The images show complex catenated DNA species involving crossovers between multiple double-stranded DNA molecules that are consistent with full catenanes. E. coli single-stranded DNA-binding protein significantly stimulated both the topoisomerase activity of Topo III alone and the DNA strand passage activity of RecQ helicase and Topo III. Titration data suggest that an intermediate of the RecQ helicase unwinding process, perhaps a RecQ helicase-DNA fork, is the target for Topo III action. Catenated DNA is the predominant product under conditions of molecular crowding; however, we also discovered that RecQ helicase and single-stranded DNA-binding protein greatly stimulated the intramolecular strand passage ("supercoiling") activity of Topo III, as revealed by changes in the linking number of uncatenated DNA. Together our results demonstrate that RecQ helicase and Topo III function together to comprise a potent and concerted single-strand DNA passage activity that can mediate both catenation-decatenation processes and changes in DNA topology.     

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.     

Identification of the SSB Binding Site on E. coli RecQ Reveals a Conserved Surface for Binding SSB's C Terminus

RecQ DNA helicases act in conjunction with heterologous partner proteins to catalyze DNA metabolic activities, including recombination initiation and stalled replication fork processing. For the prototypical Escherichia coli RecQ protein, direct interaction with single-stranded DNA-binding protein (SSB) stimulates its DNA unwinding activity. Complex formation between RecQ and SSB is mediated by the RecQ winged-helix domain, which binds the nine C-terminal-most residues of SSB, a highly conserved sequence known as the SSB-Ct element. Using nuclear magnetic resonance and mutational analyses, we identify the SSB-Ct binding pocket on E. coli RecQ. The binding site shares a striking electrostatic similarity with the previously identified SSB-Ct binding site on E. coli exonuclease I, although the SSB binding domains in the two proteins are not otherwise related structurally. Substitutions that alter RecQ residues implicated in SSB-Ct binding impair RecQ binding to SSB and SSB/DNA nucleoprotein complexes. These substitutions also diminish SSB-stimulated DNA helicase activity in the variants, although additional biochemical changes in the RecQ variants indicate a role for the winged-helix domain in helicase activity beyond SSB protein binding. Sequence changes in the SSB-Ct element are sufficient to abolish interaction with RecQ in the absence of DNA and to diminish RecQ binding and helicase activity on SSB/DNA substrates. These results support a model in which RecQ has evolved an SSB-Ct binding site on its winged-helix domain as an adaptation that aids its cellular functions on SSB/DNA nucleoprotein substrates.     

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.     

Structural and functional analyses of disease-causing missense mutations in Bloom syndrome protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by genomic instability and the early development of many types of cancer. Missense mutations have been identified in the BLM gene (encoding a RecQ helicase) in affected individuals, but the molecular mechanism and the structural basis of the effects of these mutations remain to be elucidated. We analysed five disease-causing missense mutations that are localized in the BLM helicase core region: Q672R, I841T, C878R, G891E and C901Y. The disease-causing mutants had low ATPase and helicase activities but their ATP binding abilities were normal, except for Q672, whose ATP binding activity was lower than that of the intact BLM helicase. Mutants C878R, mapping near motif IV, and G891E and C901Y, mapping in motif IV, displayed severe DNA-binding defects. We used molecular modelling to analyse these mutations. Our work provides insights into the molecular basis of BLM pathology, and reveals structural elements implicated in coupling DNA binding to ATP hydrolysis and DNA unwinding. Our findings will help to explain the mechanism underlying BLM catalysis and interpreting new BLM causing mutations identified in the future.