Helicase structure and mechanism

Structural information on helicase proteins has expanded recently beyond the DNA helicases Rep and PcrA, and the hepatitis C virus RNA helicase to include UvrB, members of the DEA(D/H)-box RNA helicase family, examples of DnaB-related helicases and RuvB. The expanding database of structures has clarified the structural 'theme and variations' that relate the different helicase families. Furthermore, information is emerging on the functions of the conserved helicase motifs and their participation in the mechanisms by which these proteins catalyze the remodeling of DNA and RNA in ATP-dependent activities.     

杆状病毒DNA解旋酶

ＤＮＡ解旋酶 (ｈｅｌｉｃａｓｅ)是ＤＮＡ复制过程中一类重要的酶 ,负责打开ＤＮＡ双链 ,并参与新生链的合成 ,在ＤＮＡ修复和重组过程中都发挥着必不可少的作用。杆状病毒ＤＮＡ解旋酶除了参与ＤＮＡ复制外 ,对于晚期基因的转录、关闭宿主蛋白质合成及决定杆状病毒宿主域方面都有重要的作用。1.杆状病毒解旋酶的结构目前已有 5种杆状病毒的ＤＮＡ解旋酶基因得到克隆并测序 ,分别是苜蓿银纹夜蛾核多角体病毒(ＡｃＭＮＰＶ) ,黄杉毒蛾核多角体病毒 (ＯｐＭＮＰＶ) ,家蚕核多角体病毒 (ＢｍＮＰＶ) ,甜菜夜蛾核多角体病毒(ＳｅＭＮＰＶ)及粉…     

Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

We have developed a system to reconstitute all of the proposed steps of Okazaki fragment processing using purified yeast proteins and model substrates. DNA polymerase δ was shown to extend an upstream fragment to displace a downstream fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of fragment processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase δ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1.Eukaryotic cellular DNA is replicated semi-conservatively in the 5′ to 3′ direction. A leading strand is synthesized by DNA polymerase ϵ in a continuous manner in the direction of opening of the replication fork (1, 2). A lagging strand is synthesized by DNA polymerase δ (pol δ)³ in the opposite direction in a discontinuous manner, producing segments called Okazaki fragments (3). These stretches of ∼150 nucleotides (nt) must be joined together to create the continuous daughter strand. DNA polymerase α/primase (pol α) initiates each fragment by synthesizing an RNA/DNA primer consisting of ∼1-nt of RNA and ∼10–20 nt of DNA (4). The sliding clamp proliferating cell nuclear antigen (PCNA) is loaded on the DNA by replication factor C (RFC). pol δ then complexes with PCNA and extends the primer. When pol δ reaches the 5′-end of the downstream Okazaki fragment, it displaces the end into a flap while continuing synthesis, a process known as strand displacement (5, 6). These flap intermediates are cleaved by nucleases to produce a nick for DNA ligase I (LigI) to seal, completing the DNA strand.In one proposed mechanism for flap processing, the only required nuclease is flap endonuclease 1 (FEN1). pol δ displaces relatively short flaps, which are cleaved by FEN1 as they are created, leaving a nick for LigI (7–9). FEN1 binds at the 5′-end of the flap and tracks down the flap cleaving only at the base (5, 10, 11). Because pol δ favors the displacement of RNA-DNA hybrids over DNA-DNA hybrids, strand displacement generally is limited to that of the initiator RNA of an Okazaki fragment (12). In addition, the tightly coordinated action of pol δ and FEN1 also tends to keep flaps short. However, biochemical reconstitution studies demonstrate that some flaps can become long (13, 14). Once these flaps reach ∼30 nt, they can be bound by the eukaryotic single strand binding protein replication protein A (RPA) (15). Binding by RPA to a flap substrate inhibits cleavage by FEN1 (16). The RPA-bound flap would then require another mechanism for proper processing.This second mechanism is proposed to utilize Dna2 (16) in addition to FEN1. Dna2 is both a 5′-3′ helicase and an endonuclease (17, 18). Like FEN1, Dna2 recognizes 5′-flap structures, binding at the 5′-end of the flap and tracking downward toward the base (19, 20). Unlike FEN1, Dna2 cleaves the flap multiple times but not all the way to the base, such that a short flap remains (20). RPA binding to a flap has been shown to stimulate Dna2 cleavage (16). Therefore, if a flap becomes long enough to bind RPA, Dna2 binds and cleaves it to a length of 5–10 nucleotides from which RPA dissociates (21). FEN1 can then enter the flap, displace the Dna2, and then cleave at the base to make the nick for ligation (16, 18, 22). The need for this mechanism may be one reason why DNA2 is an essential gene in Saccharomyces cerevisiae (23, 24). It has been proposed that, in the absence of Dna2, flaps that become long enough to bind RPA cannot be properly processed, leading to genomic instability and cell death (23).In reconstitution of Okazaki fragment processing with purified proteins, even though some flaps became long enough to bind RPA, FEN1 was very effective at cleaving essentially all of the generated flaps (13, 14). Evidently, FEN1 could engage the flaps before binding of RPA. However, these reconstitution assays did not include the 5′-3′ helicase Pif1 (25, 26). Pif1 is involved in telomeric and mitochondrial DNA maintenance (26) and was first implicated in Okazaki fragment processing from genetic studies in S. cerevisiae. Deletion of PIF1 rescued the lethality of dna2Δ, although the double mutant was still temperature-sensitive (27). The authors of this report proposed that Pif1 creates a need for Dna2 by promoting longer flaps. Further supporting this conclusion, deletion of POL32, which encodes the subunit of pol δ that interacts with PCNA, rescued the temperature sensitivity of the dna2Δpif1Δ double mutant (12, 27). Importantly, pol δ exhibited reduced strand displacement activity when POL32 was deleted (12, 28, 29). The combination of pif1Δ and pol32Δ is believed to create a situation in which virtually no long flaps are formed, eliminating the requirement for Dna2 flap cleavage (27).We recently performed reconstitution assays showing that Pif1 can assist in the creation of long flaps. Inclusion of Pif1, in the absence of RPA, increased the proportion of flaps that lengthened to ∼28–32 nt before FEN1 cleavage (14). With the addition of RPA, the appearance of these long flap cleavage products was suppressed. Evidently, Pif1 promoted such rapid flap lengthening that RPA bound some flaps before FEN1 and inhibited cleavage. The RPA-bound flaps would presumably require cleavage by Dna2 for proper processing.Only a small fraction of flaps became long with Pif1. However, there are hundreds of thousands of Okazaki fragments processed per replication cycle (30). Therefore, thousands of flaps are expected to be lengthened by Pif1 in vivo, a number significant enough that improper processing of such flaps could lead to cell death.Our goal here was to determine whether Pif1 can influence the flow of Okazaki fragments through the two proposed pathways. We first questioned whether Pif1 stimulates strand displacement synthesis by pol δ. Next, we asked whether Pif1 lengthens short flaps so that Dna2 can bind and cleave. Finally, we used a complete reconstitution system to determine whether Pif1 promotes creation of RPA-bound flaps that require cleavage by both Dna2 and FEN1 before they can be ligated. Our results suggest that Pif1 promotes the two-nuclease pathway, and reveal the mechanisms involved.     

The Minichromosome Maintenance Replicative Helicase

The eukaryotic replicative helicase, the minichromosome maintenance (MCM) complex, is composed of six distinct, but related, subunits MCM(2–7). The relationship between the sequences of the subunits indicates that they are derived from a common ancestor and indeed, present-day archaea possess a homohexameric MCM. Recent progress in the biochemical and structural studies of both eukaryal and archaeal MCM complexes are beginning to shed light on the mechanisms of action of this key component of the replisome.The minichromosome maintenance (MCM) complex subunits are members of the AAA⁺ superfamily of ATPases and thus use energy derived from cycles of ATP binding and hydrolysis to move or reorganize bound substrates. In the case of the MCM complex, the energy is harnessed to effect DNA unwinding. The AAA⁺ proteins can be classified into seven distinct clades, based on the topography of their active sites. MCMs are members of clade 7, being characterized by the presence of an additional α-helix when compared with the classical AAA⁺ fold (Iyer et al. 2004; Erzberger and Berger 2006). In addition to the AAA⁺ domain, MCMs also have an amino-terminal domain (NTD) that plays a role in higher-order structure assembly. Finally, following the AAA⁺ domain is a degenerate winged helix (wH) structure (Fig. 1). Although the archaeal MCMs possess this simple NTD–AAA⁺–wH domain architecture, many of the eukaryal MCM(2–7) subunits are embellished with amino- or carboxy-terminal extensions that play roles in the regulation or recruitment of MCM(2–7). The eukaryal MCM complex is an important target for regulatory posttranslational modifications; the nature and consequences of these modifications are dealt with in Bell and Kaguni (2013), Tanaka and Araki (2013), and Siddiqui et al. (2013). Much of what we know regarding the inner workings of the MCM helicase has been learned from structural and mechanistic studies of the simple archaeal model (Sakakibara et al. 2009). Additionally, the crystal structures of distantly related superfamily three helicases, SV40 LTAg, and the E1 helicase of bovine papilloma virus, have provided important structural frameworks for understanding the mode of action of hexameric helicases (Gai et al. 2004; Enemark and Joshua-Tor 2006, 2008). In the following, we shall describe structural and mechanistic insights derived from studies of the simpler archaeal MCMs before extending our discussion to the eukaryotic assembly.Open in a separate windowFigure 1.(A) Linear representation of a monomer of the archaeal MCM. (Gray) The central AAA⁺ domain; (white) the flanking amino-terminal domains and winged helix (wH). The position of key secondary structural elements—(Zn) zinc-binding; (ACL) allosteric communication loop; (NBH) amino-terminal β-hairpin; (EXT-HP) external β-hairpin; (H2I) helix 2 insert; and (PS1BH) pre-sensor 1 β-hairpin—are indicated above the figure and shown by colored blocks, the colors corresponding to those used in panels B and C. Key residues involved in the ATPase active site are indicated below the figure. (Orange lines) A, B, and S1, shown as orange lines are the Walker A lysine (K346), Walker B glutamate (E404), and Sensor 1 asparagine (N448), respectively, and constitute “cis”-acting residues. (Black lines) “Trans”-acting residues T1 (R331), T2 (Q423), arginine finger (R473), and Sensor 2 (R560). Numbering is from SsoMCM.(B) Structure of a monomer of SsoMCM (lacking detail of the wH domain). Secondary structure elements are labeled and colored in cartoon format, using the color scheme in panel A. (Purple spheres) The atoms of the zinc-coordinating residues; (orange spheres) the cis-acting residues; (black spheres) the trans-residues.(C) Model of a symmetric hexamer of SsoMCM. (Left) View down the central cavity of SsoMCM, looking from the carboxy-terminal face. (Right) The same hexamer rotated 90° to show a side view. The two tiers corresponding to the amino-terminal and AAA⁺ domains are indicated. The color scheme is as in panels A and B. Panels B and C were generated from PDB entry 3F9V using PyMOL (http://www.pymol.org).     

Helicase: mystery of progression

Helicases mode of unwinding the nucleic acids and translocation along single stranded nucleic acids is still a subject of great curiosity. Based on the energy transduction and electrophilic interactions, we present a model to explain the mode of action of active helicases. This model considers that both strand separation as well as translocation is active processes fueled by NTP hydrolysis. The model proposes that the translocation appears to involve creeping of helicase over the ssNA lattice rather than inchworm movement.     

Helicase motifs: the engine that powers DNA unwinding

Helicases play essential roles in nearly all DNA metabolic transactions and have been implicated in a variety of human genetic disorders. A hallmark of these enzymes is the existence of a set of highly conserved amino acid sequences termed the 'helicase motifs' that were hypothesized to be critical for helicase function. These motifs are shared by another group of enzymes involved in chromatin remodelling. Numerous structure-function studies, targeting highly conserved residues within the helicase motifs, have been instrumental in uncovering the functional significance of these regions. Recently, the results of these mutational studies were augmented by the solution of the three-dimensional crystal structure of three different helicases. The structural model for each helicase revealed that the conserved motifs are clustered together, forming a nucleotide-binding pocket and a portion of the nucleic acid binding site. This result is gratifying, as it is consistent with structure-function studies suggesting that all the conserved motifs are involved in the nucleotide hydrolysis reaction. Here, we review helicase structure-function studies in the light of the recent crystal structure reports. The current data support a model for helicase action in which the conserved motifs define an engine that powers the unwinding of duplex nucleic acids, using energy derived from nucleotide hydrolysis and conformational changes that allow the transduction of energy between the nucleotide and nucleic acid binding sites. In addition, this ATP-hydrolysing engine is apparently also associated with proteins involved in chromatin remodelling and provides the energy required to alter protein-DNA structure, rather than duplex DNA or RNA structure.     

Helicase homologues maintain cytosine methylation in plants and mammals

The Arabidopsis DDM1 gene is required for the maintenance of genomic methylation patterns but is a helicase homolog of the SWI2/SNF2 family rather than a DNA methyltransferase. Dennis et al. have shown that disruption of the mouse Lsh gene, the mammalian gene most closely related to DDM1, causes demethylation of the mouse genome. This result suggests that the mechanisms that maintain methylation patterns in the genomes of mammals and flowering plants are more conserved than previously suspected.     

Fuel Specificity of the Hepatitis C Virus NS3 Helicase

The hepatitis C virus (HCV) NS3 protein is a helicase capable of unwinding duplex RNA or DNA. This study uses a newly developed molecular-beacon-based helicase assay (MBHA) to investigate how nucleoside triphosphates (NTPs) fuel HCV helicase-catalyzed DNA unwinding. The MBHA monitors the irreversible helicase-catalyzed displacement of an oligonucleotide-bound molecular beacon so that rates of helicase translocation can be directly measured in real time. The MBHA reveals that HCV helicase unwinds DNA at different rates depending on the nature and concentration of NTPs in solution, such that the fastest reactions are observed in the presence of CTP followed by ATP, UTP, and GTP. 3′-Deoxy-NTPs generally support faster DNA unwinding, with dTTP supporting faster rates than any other canonical (d)NTP. The presence of an intact NS3 protease domain makes HCV helicase somewhat less specific than truncated NS3 bearing only its helicase region (NS3h). Various NTPs bind NS3h with similar affinities, but each NTP supports a different unwinding rate and processivity. Studies with NTP analogs reveal that specificity is determined by the nature of the Watson-Crick base-pairing region of the NTP base and the nature of the functional groups attached to the 2′ and 3′ carbons of the NTP sugar. The divalent metal bridging the NTP to NS3h also influences observed unwinding rates, with Mn²⁺ supporting about 10 times faster unwinding than Mg²⁺. Unlike Mg²⁺, Mn²⁺ does not support HCV helicase-catalyzed ATP hydrolysis in the absence of stimulating nucleic acids. Results are discussed in relation to models for how ATP might fuel the unwinding reaction.     

Helicase Loading at Chromosomal Origins of Replication

Loading of the replicative DNA helicase at origins of replication is of central importance in DNA replication. As the first of the replication fork proteins assemble at chromosomal origins of replication, the loaded helicase is required for the recruitment of the rest of the replication machinery. In this work, we review the current knowledge of helicase loading at Escherichia coli and eukaryotic origins of replication. In each case, this process requires both an origin recognition protein as well as one or more additional proteins. Comparison of these events shows intriguing similarities that suggest a similar underlying mechanism, as well as critical differences that likely reflect the distinct processes that regulate helicase loading in bacterial and eukaryotic cells.Replicative DNA helicases are ring-shaped molecules with a central cavity through which DNA passes as they unwind DNA. Their loading at replication origins is a critical and highly regulated event in chromosomal replication. The DNA helicase is the first of the replication fork proteins recruited to and loaded onto origins of replication, and the loaded helicase is required for the recruitment of the rest of the replication machinery (Remus and Diffley 2009; Kaguni 2011). Indeed, the replicative DNA helicase links the replication machinery to the parental DNA (O’Donnell 2006). In Escherichia coli cells, the DnaB replicative helicase binds to primase (DnaG) and the sliding clamp loader, which in turn binds the DNA polymerases. Although the polymerases are also linked to the template DNA by sliding clamps, when these interactions are broken, the polymerases’ association with the sliding clamp loader and the helicase keeps them at the site of replication. The interactions that tether the DNA polymerases to the eukaryotic replication fork are less clear but very likely involve direct and indirect interactions with the Mcm2–7 replicative helicase (Calzada et al. 2005).Helicase loading is carefully regulated to control the location and frequency of replication initiation. In eukaryotic cells, helicase loading is tightly restricted to the G₁ phase of the cell cycle. This constraint is a key part of the mechanisms that ensure that no origin can initiate more than once per cell cycle (Siddiqui et al. 2013; Zielke et al. 2013). In addition, the sites of eukaryotic replicative helicase loading define the potential sites of replication initiation in the cell (but not all loaded helicases are used during a given S phase [Rhind and Gilbert 2012]). Although the central regulated event in bacterial chromosome duplication is the recruitment of the ATP-bound initiator protein DnaA (Skarstad and Katayama 2013), the loading of the replicative helicase represents a key committed step during initiation.Here we will discuss the mechanism of helicase loading in bacteria and eukaryotic cells. Much of the discussion will focus on studies in the bacterium E. coli, the yeast Saccharomyces cerevisiae, and the frog Xenopus laevis, in which the events of helicase loading are best understood. Comparison of these mechanisms shows important similarities and differences between the domains of life. In both bacteria and eukaryotic cells, multiple AAA⁺ proteins use ATP binding and hydrolysis to direct helicase loading and both helicases are initially loaded in an inactive form. On the other hand, the eukaryotic helicase is loaded around double-stranded DNA (dsDNA) and as a double hexamer, whereas the bacterial helicase is loaded around single-stranded DNA (ssDNA) as a single hexamer. These distinctions are very likely due to the very different regulatory mechanisms of DNA replication in bacteria and eukaryotic cells.Before describing the process of helicase loading, it is relevant to know that the essential function of replicative helicases is to unwind the parental duplex DNA using the energy provided by the hydrolysis of a nucleoside triphosphate (Patel et al. 2011). Replicative DNA polymerases then copy each parental DNA strand to duplicate the genome. That replicative DNA helicases are hexameric (and sometimes heptameric) structures that have a positively charged, central channel provides a framework for how these molecular machines work. Several models that describe the mechanism of unwinding have been considered. One is the steric or strand-exclusion model, in which the helicase excludes one strand of DNA while the other passes through the central cavity as the enzyme moves. Because these enzymes can bind and translocate on duplex DNA without unwinding (Kaplan et al. 2003), two additional models have been proposed. In the ploughshare model, duplex DNA enters the central cavity and exits as unwound DNA by virtue of a domain/protein that acts as a ploughshare or pin, which disrupts the hydrogen bonds of DNA as it is pumped through the enzyme (reviewed in Takahashi et al. 2005). A second model, the DNA-pumping model, was proposed on the basis of the double-hexameric forms of SV40, Mcm2–7, and other replicative helicases (Mastrangelo et al. 1989; Remus et al. 2009). This model proposes that the two helicases pump duplex DNA toward one another, resulting in torsional strain that forces the two strands apart, at which point they exit the central channel as two ssDNA loops. Current evidence supports the steric exclusion model (Jezewska et al. 1998b; Kaplan 2000; Galletto et al. 2004; Fu et al. 2011). Of interest, the E. coli enzyme moves in the 5′-to-3′ direction relative to the engaged ssDNA that passes through the central cavity, whereas archaeal and eukaryotic enzymes move in the 3′-to-5′ direction.

Table 1.

Replicative DNA helicases of free-living organisms are hexameric

Mutations in DDX59 Implicate RNA Helicase in the Pathogenesis of Orofaciodigital Syndrome

Orofaciodigital syndrome (OFD) is a recognized clinical entity with core defining features in the mouth, face, and digits, in addition to various other features that have been proposed to define distinct subtypes. The three genes linked to OFD—OFD1, TMEM216, and TCTN3—play a role in ciliary biology, a finding consistent with the clinical overlap between OFD and other ciliopathies. Most autosomal-recessive cases of OFD, however, remain undefined genetically. In two multiplex consanguineous Arab families affected by OFD, we identified a tight linkage interval in chromosomal region 1q32.1. Exome sequencing revealed a different homozygous variant in DDX59 in each of the two families, and at least one of the two variants was accompanied by marked reduction in the level of DDX59. DDX59 encodes a relatively uncharacterized member of the DEAD-box-containing RNA helicase family of proteins, which are known to play a critical role in all aspects of RNA metabolism. We show that Ddx59 is highly enriched in its expression in the developing murine palate and limb buds. At the cellular level, we show that DDX59 is localized dynamically to the nucleus and the cytoplasm. Consistent with the absence of DDX59 representation in ciliome databases and our demonstration of its lack of ciliary localization, ciliogenesis appears to be intact in mutant fibroblasts but ciliary signaling appears to be impaired. Our data strongly implicate this RNA helicase family member in the pathogenesis of OFD, although the causal mechanism remains unclear.     

Regulation of UvrD Helicase Activity by MutL

Escherichia coli UvrD is a superfamily 1 helicase/translocase involved in multiple DNA metabolic processes including methyl-directed mismatch DNA repair. Although a UvrD monomer can translocate along single-stranded DNA, a UvrD dimer is needed for processive helicase activity in vitro. E. coli MutL, a regulatory protein involved in methyl-directed mismatch repair, stimulates UvrD helicase activity; however, the mechanism is not well understood. Using single-molecule fluorescence and ensemble approaches, we find that a single MutL dimer can activate latent UvrD monomer helicase activity. However, we also find that MutL stimulates UvrD dimer helicase activity. We further find that MutL enhances the DNA-unwinding processivity of UvrD. Hence, MutL acts as a processivity factor by binding to and presumably moving along with UvrD to facilitate DNA unwinding.