Repetitive shuttling of a motor protein on DNA

Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3' to 5' direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates.     

ATP-induced helicase slippage reveals highly coordinated subunits

Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination. Bacteriophage T7 gene product 4 is a model hexameric helicase that has been observed to use dTTP, but not ATP, to unwind double-stranded (ds)DNA as it translocates from 5' to 3' along single-stranded (ss)DNA. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was not observed with dTTP alone and was greatly reduced when ATP solution was supplemented with a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. We found that T7 helicase binds and hydrolyses ATP and dTTP by competitive kinetics such that the unwinding rate is dictated simply by their respective maximum rates V(max), Michaelis constants K(M) and concentrations. In contrast, processivity does not follow a simple competitive behaviour and shows a cooperative dependence on nucleotide concentrations. This does not agree with an uncoordinated mechanism where each subunit functions independently, but supports a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide-ligated and thus they interact with the DNA to ensure processivity. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.     

Translocation step size and mechanism of the RecBC DNA helicase

DNA helicases are ubiquitous enzymes that unwind double-stranded DNA. They are a diverse group of proteins that move in a linear fashion along a one-dimensional polymer lattice--DNA--by using a mechanism that couples nucleoside triphosphate hydrolysis to both translocation and double-stranded DNA unwinding to produce separate strands of DNA. The RecBC enzyme is a processive DNA helicase that functions in homologous recombination in Escherichia coli; it unwinds up to 6,250 base pairs per binding event and hydrolyses slightly more than one ATP molecule per base pair unwound. Here we show, by using a series of gapped oligonucleotide substrates, that this enzyme translocates along only one strand of duplex DNA in the 3'-->5' direction. The translocating enzyme will traverse, or 'step' across, single-stranded DNA gaps in defined steps that are 23 (+/-2) nucleotides in length. This step is much larger than the amount of double-stranded DNA that can be unwound using the free energy derived from hydrolysis of one molecule of ATP, implying that translocation and DNA unwinding are separate events. We propose that the RecBC enzyme both translocates and unwinds by a quantized, two-step, inchworm-like mechanism that may have parallels for translocation by other linear motor proteins.     

RecBCD enzyme is a bipolar DNA helicase

Escherichia coli RecBCD is a heterotrimeric helicase/nuclease that catalyses a complex reaction in which double-strand breaks in DNA are processed for repair by homologous recombination. For some time it has been clear that the RecB subunit possesses a 3' --> 5' DNA helicase activity, which was thought to drive DNA translocation and unwinding in the RecBCD holoenzyme. Here we show that purified RecD protein is also a DNA helicase, but one that possesses a 5' --> 3' polarity. We also show that the RecB and RecD helicases are both active in intact RecBCD, because the enzyme remains capable of processive DNA unwinding when either of these subunits is inactivated by mutation. These findings point to a bipolar translocation model for RecBCD in which the two DNA helicases are complementary, travelling with opposite polarities, but in the same direction, on each strand of the antiparallel DNA duplex. This bipolar motor organization helps to explain various biochemical properties of RecBCD, notably its exceptionally high speed and processivity, and offers a mechanistic insight into aspects of RecBCD function.     

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.     

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.     

Structural basis of RNA recognition and activation by innate immune receptor RIG-I

Retinoic-acid-inducible gene-I (RIG-I; also known as DDX58) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds)RNA with or without a 5'-triphosphate (ppp), by single-stranded RNA marked by a 5'-ppp and by polyuridine sequences. Upon binding to such PAMP motifs, RIG-I initiates a signalling cascade that induces innate immune defences and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signalling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases and cancer. The helicase and repressor domains (RD) of RIG-I recognize dsRNA and 5'-ppp RNA to activate the two amino-terminal caspase recruitment domains (CARDs) for signalling. Here, to understand the synergy between the helicase and the RD for RNA binding, and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP analogue. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands using previously uncharacterized motifs to recognize dsRNA. Small-angle X-ray scattering, limited proteolysis and differential scanning fluorimetry indicate that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full-length RIG-I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including RNA interference and DNA repair, which utilize homologous helicase domains within DICER and FANCM.     

Dynamics of ATP-dependent chromatin assembly by ACF

The assembly of DNA into chromatin is a critical step in the replication and repair of the eukaryotic genome. It has been known for nearly 20 years that chromatin assembly is an ATP-dependent process. ATP-dependent chromatin-assembly factor (ACF) uses the energy of ATP hydrolysis for the deposition of histones into periodic nucleosome arrays, and the ISWI subunit of ACF is an ATPase that is related to helicases. Here we show that ACF becomes committed to the DNA template upon initiation of chromatin assembly. We also observed that ACF assembles nucleosomes in localized arrays, rather than randomly distributing them. By using a purified ACF-dependent system for chromatin assembly, we found that ACF hydrolyses about 2#150;4 molecules of ATP per base pair in the assembly of nucleosomes. This level of ATP hydrolysis is similar to that used by DNA helicases for the unwinding of DNA. These results suggest that a tracking mechanism exists in which ACF assembles chromatin as an ATP-driven DNA-translocating motor. Moreover, this proposed mechanism for ACF may be relevant to the function of other chromatin-remodelling factors that contain ISWI subunits.     

Kinetic study of the DNA annealing properties of RECQ5β helicase

RecQ family helicases are critical for maintaining genomic integrity. Many RecQ family helicases not only unwind duplex, and other more complicated DNA structures, but also possess, interestingly, DNA annealing (strand pairing) activity. Here, we systematically investigated the DNA annealing properties of RECQ5β by measuring DNA annealing kinetics, equilibrium DNA binding, and kinetics of dissociation from ssDNA. RECQ5β catalyzed DNA annealing most efficiently when the enzyme molecules covered approximately 40%-50% of the DNA strand, in the absence or presence of different nucleotide cofactors (AMPPNP, ATPγS, or ADP) under our buffer conditions. A comparative study with RECQ5β1-662 confirmed that the C-terminal region of RECQ5β was essential for its high DNA annealing activity. These results contribute to our understanding of the mechanism of DNA annealing catalyzed by RecQ family helicases.     

Periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase

The NS3 helicase is essential for cytoplasmic RNA replication by the hepatitis C virus, and it is a representative member of helicase superfamily 2 (SF2). NS3 is an important model system for understanding unwinding activities of DExH/D proteins, and it has been the subject of extensive structural and mutational analyses. Despite intense interest in NS3, the molecular and kinetic mechanisms for RNA unwinding by this helicase have remained obscure. We have developed a combinatorial, time-resolved approach for monitoring the microscopic behaviour of a helicase at each nucleotide of a duplex substrate. By applying this analysis to NS3, we have independently established the 'physical' and 'kinetic' step size for unwinding of RNA (18 base pairs, in each case), which we relate to the stoichiometry of the functional, translocating species. Having obtained microscopic unwinding rate constants at each position along the duplex, we demonstrate that NS3 unwinds RNA through a highly coordinated cycle of fast ripping and local pausing that occurs with regular spacing along the duplex substrate, much like the stepping behaviour of cytoskeletal motor proteins.     

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.     

Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen

The oncoprotein large tumour antigen (LTag) is encoded by the DNA tumour virus simian virus 40. LTag transforms cells and induces tumours in animals by altering the functions of tumour suppressors (including pRB and p53) and other key cellular proteins. LTag is also a molecular machine that distorts/melts the replication origin of the viral genome and unwinds duplex DNA. LTag therefore seems to be a functional homologue of the eukaryotic minichromosome maintenance (MCM) complex. Here we present the X-ray structure of a hexameric LTag with DNA helicase activity. The structure identifies the p53-binding surface and reveals the structural basis of hexamerization. The hexamer contains a long, positively charged channel with an unusually large central chamber that binds both single-stranded and double-stranded DNA. The hexamer organizes into two tiers that can potentially rotate relative to each other through connecting alpha-helices to expand/constrict the channel, producing an 'iris' effect that could be used for distorting or melting the origin and unwinding DNA at the replication fork.     

Direct observation of the mechanochemical coupling in myosin Va during processive movement

Myosin Va transports intracellular cargoes along actin filaments in cells. This processive, two-headed motor takes multiple 36-nm steps in which the two heads swing forward alternately towards the barbed end of actin driven by ATP hydrolysis. The ability of myosin Va to move processively is a function of its long lever arm, the high duty ratio of its kinetic cycle and the gating of the kinetics between the two heads such that ADP release from the lead head is greatly retarded. Mechanical studies at the multiple- and the single-molecule level suggest that there is tight coupling (that is, one ATP is hydrolysed per power stroke), but this has not been directly demonstrated. We therefore investigated the coordination between the ATPase mechanism of the two heads of myosin Va and directly visualized the binding and dissociation of single fluorescently labelled nucleotide molecules, while simultaneously observing the stepping motion of the fluorescently labelled myosin Va as it moved along an actin filament. Here we show that preferential ADP dissociation from the trail head of mouse myosin Va is followed by ATP binding and a synchronous 36-nm step. Even at low ATP concentrations, the myosin Va molecule retained at least one nucleotide (ADP in the lead head position) when moving. Thus, we directly demonstrate tight coupling between myosin Va movement and the binding and dissociation of nucleotide by simultaneously imaging with near nanometre precision.     

PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome.

The assembly of the spliceosome is an ATP-dependent process. The splicing factor PRP16 contains variations of several motifs that define the eIF-4A-like ATP-dependent RNA helicase family. The protein has now been purified and shown to exhibit RNA-dependent ATPase activity. PRP16 is required specifically for the second catalytic step of the splicing reaction in vitro. This function requires ATP binding and/or hydrolysis, which appears to be concomitant with release of the protein from the spliceosome. PRP16 may be the prototype for a set of splicing factors which use ATP to drive a cycle of conformational changes.     

Mg2+对Bloom综合症解旋酶与G4DNA结合的影响研究

利用荧光偏振技术检测了Mg2+对G4DNA、BLM-G4DNA复合物和BLM642-1290解旋酶与G4DNA结合的影响.结果表明,G4DNA荧光偏振值随着Mg2+浓度的增加而增加(P＜0.01);BLM-G4DNA复合物的荧光偏振值随着Mg2+浓度的增加出现下降—升高—下降的变化趋势(P＜0.01);G4DNA与BLM642-1290解旋酶结合的荧光偏振值随着Mg2+浓度的增加而逐渐下降(P＜0.01);分析不同Mg2+浓度下两种分子结合的Kd值,发现Mg2+浓度为3.0 mmol/L时,BLM642-1290解旋酶与G4DNA最容易结合,表明适量Mg2+浓度会促进BLM642-1290与G4DNA的结合,但会引起两种分子结合的形状、流动性和电荷等性质的改变.这些结果可为进一步研究BLM解旋酶对G4DNA的作用机理提供相关资料.     

The DExH protein NPH-II is a processive and directional motor for unwinding RNA

All aspects of cellular RNA metabolism and processing involve DExH/D proteins, which are a family of enzymes that unwind or manipulate RNA in an ATP-dependent fashion. DExH/D proteins are also essential for the replication of many viruses, and therefore provide targets for the development of therapeutics. All DExH/D proteins characterized to date hydrolyse nucleoside triphosphates and, in most cases, this activity is stimulated by the addition of RNA or DNA. Several members of the family unwind RNA duplexes in an NTP-dependent fashion in vitro; therefore it has been proposed that DExH/D proteins couple NTP hydrolysis to RNA conformational change in complex macromolecular assemblies. Despite the central role of DExH/D proteins, their mechanism of RNA helicase activity remains unknown. Here we show that the DExH protein NPH-II unwinds RNA duplexes in a processive, unidirectional fashion with a step size of roughly one-half helix turn. We show that there is a quantitative connection between ATP utilization and helicase processivity, thereby providing direct evidence that DExH/D proteins can function as molecular motors on RNA.     

Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures

The RecA family of ATPases mediates homologous recombination, a reaction essential for maintaining genomic integrity and for generating genetic diversity. RecA, ATP and single-stranded DNA (ssDNA) form a helical filament that binds to double-stranded DNA (dsDNA), searches for homology, and then catalyses the exchange of the complementary strand, producing a new heteroduplex. Here we have solved the crystal structures of the Escherichia coli RecA-ssDNA and RecA-heteroduplex filaments. They show that ssDNA and ATP bind to RecA-RecA interfaces cooperatively, explaining the ATP dependency of DNA binding. The ATP gamma-phosphate is sensed across the RecA-RecA interface by two lysine residues that also stimulate ATP hydrolysis, providing a mechanism for DNA release. The DNA is underwound and stretched globally, but locally it adopts a B-DNA-like conformation that restricts the homology search to Watson-Crick-type base pairing. The complementary strand interacts primarily through base pairing, making heteroduplex formation strictly dependent on complementarity. The underwound, stretched filament conformation probably evolved to destabilize the donor duplex, freeing the complementary strand for homology sampling.     

Structure of the recA protein-ADP complex.

The recA protein catalyses the ATP-driven homologous pairing and strand exchange of DNA molecules. It is an allosteric enzyme: the ATPase activity is DNA-dependent, and ATP-bound recA protein has a high affinity for DNA, whereas the ADP-bound form has a low affinity. In the absence of ATP hydrolysis, recA protein can still promote homologous pairing, apparently through the formation of a triple-stranded intermediate. The exact role of ATP hydrolysis is not clear, but it presumably drives the triplex intermediate towards products. Here we determine the position of bound ADP diffused into the recA crystal. We show that only the phosphates are bound in the same way as in other NTPases containing the G/AXXXXGKT/S motif. We propose that recA protein may change its conformation upon ATP hydrolysis in a manner analogous to one such protein, the p21 protein from the ras oncogene. A model is presented to account for the allosteric stimulation of DNA binding by ATP. The mechanism by which nucleoside triphosphate hydrolysis is coupled to the binding of another ligand in recA protein and p21 may be typical of the large class of NTPases containing this conserved motif.     

Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks

RecBCD is a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break. RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site). The nuclease activity is then attenuated and RecBCD loads RecA onto the 3' tail of the DNA. Here we present the crystal structure of RecBCD bound to a DNA substrate. In this initiation complex, the DNA duplex has been split across the RecC subunit to create a fork with the separated strands each heading towards different helicase motor subunits. The strands pass along tunnels within the complex, both emerging adjacent to the nuclease domain of RecB. Passage of the 3' tail through one of these tunnels provides a mechanism for the recognition of a Chi sequence by RecC within the context of double-stranded DNA. Gating of this tunnel suggests how nuclease activity might be regulated.     

Electron cryo-microscopy shows how strong binding of myosin to actin releases nucleotide

Muscle contraction involves the cyclic interaction of the myosin cross-bridges with the actin filament, which is coupled to steps in the hydrolysis of ATP. While bound to actin each cross-bridge undergoes a conformational change, often referred to as the "power stroke", which moves the actin filament past the myosin filaments; this is associated with the release of the products of ATP hydrolysis and a stronger binding of myosin to actin. The association of a new ATP molecule weakens the binding again, and the attached cross-bridge rapidly dissociates from actin. The nucleotide is then hydrolysed, the conformational change reverses, and the myosin cross-bridge reattaches to actin. X-ray crystallography has determined the structural basis of the power stroke, but it is still not clear why the binding of actin weakens that of the nucleotide and vice versa. Here we describe, by fitting atomic models of actin and the myosin cross-bridge into high-resolution electron cryo-microscopy three-dimensional reconstructions, the molecular basis of this linkage. The closing of the actin-binding cleft when actin binds is structurally coupled to the opening of the nucleotide-binding pocket.