Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase

Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid, and are involved in several human diseases. Some function as hexameric rings, but the functional form of non-hexameric helicases has been debated. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays--which do not interfere with biological activity--to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions ('stalls') that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein-DNA interactions.     

DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase

Helicases are molecular motors that use the energy of nucleoside 5'-triphosphate (NTP) hydrolysis to translocate along a nucleic acid strand and catalyse reactions such as DNA unwinding. The ring-shaped helicase of bacteriophage T7 translocates along single-stranded (ss)DNA at a speed of 130 bases per second; however, T7 helicase slows down nearly tenfold when unwinding the strands of duplex DNA. Here, we report that T7 DNA polymerase, which is unable to catalyse strand displacement DNA synthesis by itself, can increase the unwinding rate to 114 base pairs per second, bringing the helicase up to similar speeds compared to its translocation along ssDNA. The helicase rate of stimulation depends upon the DNA synthesis rate and does not rely on specific interactions between T7 DNA polymerase and the carboxy-terminal residues of T7 helicase. Efficient duplex DNA synthesis is achieved only by the combined action of the helicase and polymerase. The strand displacement DNA synthesis by the DNA polymerase depends on the unwinding activity of the helicase, which provides ssDNA template. The rapid trapping of the ssDNA bases by the DNA synthesis activity of the polymerase in turn drives the helicase to move forward through duplex DNA at speeds similar to those observed along ssDNA.     

DNA helicase Srs2 disrupts the Rad51 presynaptic filament

Mutations in the Saccharomyces cerevisiae gene SRS2 result in the yeast's sensitivity to genotoxic agents, failure to recover or adapt from DNA damage checkpoint-mediated cell cycle arrest, slow growth, chromosome loss, and hyper-recombination. Furthermore, double mutant strains, with mutations in DNA helicase genes SRS2 and SGS1, show low viability that can be overcome by inactivating recombination, implying that untimely recombination is the cause of growth impairment. Here we clarify the role of SRS2 in recombination modulation by purifying its encoded product and examining its interactions with the Rad51 recombinase. Srs2 has a robust ATPase activity that is dependent on single-stranded DNA (ssDNA) and binds Rad51, but the addition of a catalytic quantity of Srs2 to Rad51-mediated recombination reactions causes severe inhibition of these reactions. We show that Srs2 acts by dislodging Rad51 from ssDNA. Thus, the attenuation of recombination efficiency by Srs2 stems primarily from its ability to dismantle the Rad51 presynaptic filament efficiently. Our findings have implications for the basis of Bloom's and Werner's syndromes, which are caused by mutations in DNA helicases and are characterized by increased frequencies of recombination and a predisposition to cancers and accelerated ageing.     

Stimulation of protein-directed strand exchange by a DNA helicase

The protein-mediated exchange of strands between a DNA double helix and a homologous DNA single strand involves both synapsis and branch migration, which are two important aspects of any general recombination reaction. Purified DNA-dependent ATPases from Escherichia coli (recA protein), Ustilago (rec 1 protein) and phage T4 (uvsX protein) have been shown to drive both synapsis and branch migration in vitro. The T4 gene 32 protein is a helix-destabilizing protein that greatly stimulates uvsX-protein-catalysed synapsis, and the E. coli SSB (single-strand binding) protein stimulates the analogous recA-protein-mediated reaction to a lesser degree. One suspects that several other proteins also play a role in the strand exchange process. For example, a DNA helicase could in principle accelerate branch migration rates by helping to melt the helix at the branch point. The T4 dda protein is a DNA helicase that is required to move the T4 replication fork past DNA template-bound proteins in vitro. Previously, we have shown that the dda protein binds to a column that contains immobilized T4 uvsX protein. We show here that this helicase specifically stimulates the branch migration reaction that the uvsX protein catalyses as a central part of the genetic recombination process in a T4 bacteriophage-infected cell.     

Mechanism of DNA translocation in a replicative hexameric helicase

The E1 protein of papillomavirus is a hexameric ring helicase belonging to the AAA + family. The mechanism that couples the ATP cycle to DNA translocation has been unclear. Here we present the crystal structure of the E1 hexamer with single-stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. This structure demonstrates that only one strand of DNA passes through the hexamer channel and that the DNA-binding hairpins of each subunit form a spiral 'staircase' that sequentially tracks the oligonucleotide backbone. Consecutively grouped ATP, ADP and apo configurations correlate with the height of the hairpin, suggesting a straightforward DNA translocation mechanism. Each subunit sequentially progresses through ATP, ADP and apo states while the associated DNA-binding hairpin travels from the top staircase position to the bottom, escorting one nucleotide of single-stranded DNA through the channel. These events permute sequentially around the ring from one subunit to the next.     

一种新的可变步长LMS算法研究

在分析基本最小均方误差算法(LMS)和归一化最小均方误差算法(NLMS)的基础上,提出了一种新的可变步长LMS算法(NVLMS)和它的改进算法(MNVLMS).仿真结果显示,NVLMS算法对于平稳过程中的滤波器,能获得较快的收敛速度和较小的稳态误差.在非平稳环境下,MNVLMS算法在减少算法复杂度的情况下能获得和NLMS算法一样的收敛速度和稳态误差.     

一种变步长LMS算法及其Matlab仿真

对变步长自适应滤波算法进行了讨论，对VS—LMS算法进行了改进，建立了步长因子μ与误差信号e（n）之间另一种新的非线性函数关系．理论分析和计算机仿真结果表明，该关系不仅具有原有算法收敛速度快的优点，而且在低信噪比环境下比原有算法具有更好的抗噪声性能．     

Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules

Major pathways of recombinational DNA repair in Escherichia coli require the RecBCD protein--a heterotrimeric, ATP-driven, DNA translocating motor enzyme. RecBCD combines a highly processive and exceptionally fast helicase (DNA-unwinding) activity with a strand-specific nuclease (DNA-cleaving) activity (refs 1, 2 and references therein). Recognition of the DNA sequence 'chi' (5'-GCTGGTGG-3') switches the polarity of DNA cleavage and stimulates recombination at nearby sequences in vivo. Here we attach microscopic polystyrene beads to biotin-tagged RecD protein subunits and use tethered-particle light microscopy to observe translocation of single RecBCD molecules (with a precision of up to approximately 30 nm at 2 Hz) and to examine the mechanism by which chi modifies enzyme activity. Observed translocation is unidirectional, with each molecule moving at a constant velocity corresponding to the population-average DNA unwinding rate. These observations place strong constraints on possible movement mechanisms. Bead release at chi is negligible, showing that the activity modification at chi does not require ejection of the RecD subunit from the enzyme as previously proposed; modification may occur through an unusual, pure conformational switch mechanism.     

基于OPNET的BotNet最优步长传播仿真

僵尸网络(BotNet)主要采用蠕虫扫描的方式进行传播,传统的蠕虫传播策略主要在扩散效率和扫描准确性上进行折衷,共性缺点是存在对同一主机重复扫描和网络之间交叉扫描的严重问题,会对互联网产生严重的流量冲击,降低BotNet的隐蔽性。为解决这一共性问题,根据互联网的无尺度特性,在对比传统网络蠕虫传播策略优缺点的基础上,结合前向神经网络现有的BP学习算法对BotNet传播进行了分析,从理论上提出一种计算BP近似最优步长的算法,并通过OPNET建立传播攻击模型进行了仿真验证。结果表明,该算法有效地提高了BotNet在无尺度网络中的传播性能。     

一种新的变步长LMS自适应滤波算法及其仿真

针对变步长自适应滤波算法收敛速度和稳态误差相矛盾的不足,建立了步长μ(n)与误差信号e(n)之间的一种新的非线性函数关系。该函数具有初始阶段和未知系统时变阶段步长自动增大而稳态时步长很小的特点,且能克服输入端不相关噪声对步长μ(n)的影响。由此函数,得出了一种新的变步长自适应算法,理论分析和计算机仿真结果表明该算法的性能优于文中所述其他算法。     

一种新的变步长LMS自适应滤波算法及其仿真

针对变步长自适应滤波算法收敛速度和稳态误差相矛盾的不足,建立了步长μ(n)与误差信号e(n)之间的一种新的非线性函数关系.该函数具有初始阶段和未知系统时变阶段步长自动增大而稳态时步长很小的特点,且能克服输入端不相关噪声对步长μ(n)的影响.由此函数,得出了一种新的变步长自适应算法,理论分析和计算机仿真结果表明该算法的性能优于文中所述其他算法.     

RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP

Helicases are a ubiquitous class of enzymes involved in nearly all aspects of DNA and RNA metabolism. Despite recent progress in understanding their mechanism of action, limited resolution has left inaccessible the detailed mechanisms by which these enzymes couple the rearrangement of nucleic acid structures to the binding and hydrolysis of ATP. Observing individual mechanistic cycles of these motor proteins is central to understanding their cellular functions. Here we follow in real time, at a resolution of two base pairs and 20 ms, the RNA translocation and unwinding cycles of a hepatitis C virus helicase (NS3) monomer. NS3 is a representative superfamily-2 helicase essential for viral replication, and therefore a potentially important drug target. We show that the cyclic movement of NS3 is coordinated by ATP in discrete steps of 11 +/- 3 base pairs, and that actual unwinding occurs in rapid smaller substeps of 3.6 +/- 1.3 base pairs, also triggered by ATP binding, indicating that NS3 might move like an inchworm. This ATP-coupling mechanism is likely to be applicable to other non-hexameric helicases involved in many essential cellular functions. The assay developed here should be useful in investigating a broad range of nucleic acid translocation motors.     

AFM观察小白鼠心肌和肝核DNA片段的基因移位

用原子力显微镜(简称AFM)直接观察、体外表达和体外转录等实验技术组合,观察到了心肌和肝的核DNA片段的基因;用磷酸缓冲液稀释,并用开关蛋白质等活性因子使其部分解离,促使核基因在核DNA片段内或核DNA片段间静态或动态移位,得到了对应核DNA片段中的相关基因,如LDH/DNA体外表达活性变化的LDH同功酶酶谱图。基因静态或动态移位均表达活性降低,且基因移位程度与其对应基因活性降低程度呈正相关性。展示了未来运用AFM和体外表达等实验技术组合研究核DNA片段的基因移位和对应基因突变机制的前景。     

RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity

Helicases are molecular motors that move along and unwind double-stranded nucleic acids. RecBCD enzyme is a complex helicase and nuclease, essential for the major pathway of homologous recombination and DNA repair in Escherichia coli. It has sets of helicase motifs in both RecB and RecD, two of its three subunits. This rapid, highly processive enzyme unwinds DNA in an unusual manner: the 5'-ended strand forms a long single-stranded tail, whereas the 3'-ended strand forms an ever-growing single-stranded loop and short single-stranded tail. Here we show by electron microscopy of individual molecules that RecD is a fast helicase acting on the 5'-ended strand and RecB is a slow helicase acting on the 3'-ended strand on which the single-stranded loop accumulates. Mutational inactivation of the helicase domain in RecB or in RecD, or removal of the RecD subunit, altered the rates of unwinding or the types of structure produced, or both. This dual-helicase mechanism explains how the looped recombination intermediates are generated and may serve as a general model for highly processive travelling machines with two active motors, such as other helicases and kinesins.     

基于自适应步长和莱维飞行策略的改进狼群算法

群智能启发算法在解决大规模分布式问题方面有许多优势。针对传统狼群算法易陷入局部最优和精度不高等缺陷,笔者在分析狼群特点的基础上,提出一种基于自适应性步长和莱维飞行搜索策略的改进狼群算法。首先,通过自适应步长的合理变化,提高搜索精度;其次,采用莱维飞行的搜索策略,在算法后期扩大搜索范围,提高算法的全局搜索能力。最后,为了验证该算法性能,通过仿真实验和实际案例进行了测试,与其他改进方法进行比较。测试结果表明,所提出的改进狼群算法在收敛速度、精度及稳定性方面都有明显优势。     

接触式台阶测量仪弹性支承机构的仿真设计

为合理设计接触式台阶测量仪 ,建立了它的简化数学模型 ,经理论计算 ,结果与理论值有较大误差。通过有限元分析软件、采用 8节点 6面体单元计算模型对机构进行数字仿真和分析。触针允许最大测量力为 8.2 6 7N,测量力变化率为 33.8N /m ,片簧回转中心偏移量约为 1μm ,可满足高精度接触式台阶测量仪的设计要求     

均方偏差分析的多态可变步长LMS算法

最小均方（least mean square,LMS）算法在时变信道的最小稳态均方偏差（mean square deviation,MSD）由输入功率、噪声功率、随机扰动信号功率以及滤波器长度共同决定。为达到系统中最小的MSD值,传统的LMS算法存在有迭代次数较多和收敛速度慢等问题,提出了一种多态可变步长最小均方（multi-state variable step size least mean square,MVSS-LMS）算法。该算法通过添加暂态递减步长作为过渡,实现以更快的收敛速度达到系统中最小的MSD值。理论分析与仿真结果表明,与目前最新的Prob-LMS算法相比,所提算法在时变信道以及突变信道都具有更快的收敛速度和更低的MSD值,且算法的复杂度更低。     

孤子在线性色散渐减光纤中压缩时自适应步长的研究

本文根据光孤子在线性色散渐减光纤中压缩时的演化规律,提出了步长随光纤中的色散长度和非线性长度的乘积变化的自适应步长分步傅里叶变换法.与定步长分步傅里叶变换法比较,同样的步数模拟,自适应步长分步傅里叶变换法可以减小引入的误差,模拟结果更准确.