Structure and Mechanisms of SF1 DNA Helicases

Abstract

Superfamily I is a large and diverse group of monomeric and dimeric helicases defined by a set of conserved sequence motifs. Members of this class are involved in essential processes in both DNA and RNA metabolism in all organisms. In addition to conserved amino acid sequences, they also share a common structure containing two RecA-like motifs involved in ATP binding and hydrolysis and nucleic acid binding and unwinding. Unwinding is facilitated by a “pin” structure which serves to split the incoming duplex. This activity has been measured using both ensemble and single-molecule conditions. SF1 helicase activity is modulated through interactions with other proteins.

Introduction

Helicases are molecular motor proteins present in viruses, bacteria, and eukaryotes [1, 2]. They harness the chemical energy of ATP hydrolysis to break the energetically stable hydrogen bonding between the duplex DNA. By doing so, helicases allow access to the genetic information locked in the duplex DNA. Helicases participate in various aspects of nucleic acid metabolism such as DNA replication, recombination, repair, transcription, translation, and splicing of RNA transcripts [3–13].

Mutations in helicase genes involved in DNA repair processes have been linked to numerous human diseases [14–17] in which genomic instability, immunodeficiency, mental retardation, premature aging, and predisposition to cancer are common features [14, 15, 18–21]. Some of the diseases caused by defective helicases are xeroderma pigmentosum, Cockayne Syndrome, trichothiodystrophy, Werner’s syndrome, Bloom’s syndrome, and alpha-thalassemia mental retardation on the X chromosome [22–30]. A mutation in superfamily 1 (SF1) helicase Pif1 results in breast cancer predisposition [31]. Mutations in SetX helicase, involved in RNA splicing and termination, cause juvenile amyotrophic lateral sclerosis [32] and ataxia-ocular apraxia 2 [33], while mutations in IGHMBP2 (Smubp2), involved in translation [34], result in distal spinal muscular atrophy [35]. The diverse disease abnormalities caused by defective helicases suggest that multiple aspects of DNA and RNA metabolism are affected [18].

In some aggressive cancers, the activity of helicases in DNA repair reduces the efficacy of anticancer agents, because many of these agents are targeted to DNA. Studies have shown that the efficacy of chemotherapeutic agents could be increased by administering drugs that target helicases along with the anticancer drugs [36]. Helicases encoded by herpes simplex virus [37–39], West Nile virus, dengue virus, and hepatitis C virus [40] are targets for antiviral drug development [38, 41]. Some bacterial helicases such as Rep from Legionella pneumophilia are required for infection of mammalian cells [42]. The importance of helicases in the fundamental aspects of nucleic acid metabolism and the association of human, bacterial, and viral helicases in human diseases makes the study of helicases critical. This also makes it essential to understand the mechanisms by which helicases perform different biochemical functions so that the relationship between mutations and specific disease states can be understood at the molecular level.

Functions of Helicases

Unwinding of duplex or structured nucleic acids by helicases provides the ssNA intermediates required for metabolism of DNA and RNA [43–45]. In vivo, DNA unwinding is coupled to the action of many proteins such as primases, ssDNA-binding proteins, polymerases, and other factors depending on the functions of a particular helicase. Many of the biological functions of various helicases are listed in Table 2.1. Helicases are implicated in processes ranging from replication to translation [8, 10–12, 46–49] and also in ATP-dependent chromatin remodeling [50, 51]. Replicative helicases deal with the process of nucleic acid replication (initiation, elongation, and termination). Helicases play an important role in DNA repair, as these are frequently the first proteins that encounter DNA damage [13, 19, 52–54]. During DNA repair, the damaged area on the DNA has to be unwound before repair can proceed as most DNA repair processes require ssDNA. Helicases play roles in both initiation and branch migration during recombination [21, 25, 28, 55–60].

Table 2.1.

Biological functions carried out by various helicases

Biological function	Helicase
Replicative helicases	PcrA1, RepA, UvrD, Dda, HSV UL5, HSV UL9, DnaB, PriA, T7gp4A and 4B, T4gp41, SV40, TAG, Polyoma TAG, BPV E1, MCM 4/6/7, Dna2, FFA-1, RecD, TraI, NS3, RecQL4
Repair helicases	UvrD, UvrAB, PcrA, Rad3, helicase E, XPD, XPB, Dna2, RecD2, BACH1, HDH II, RecQ, WRN, Rtel1, BLM, RuvB, Mph 1, CHD4
Recombination helicases	RecBCD, RecG, RecQ, RuvAB, PriA, UvrD, T4 UvsW, HDH II, HDH IV, WRN, Tra I, Rho, PDH65, BLM, Srs2, Sgs1, Rtel1
Other functions of helicases
Transcription	SWI2, SNF2, TFIIH, Rho, Factor 2, TRCF, RecQL5, ERCC6/RAD26
Translation	HSV UL5, eIF4A, RHA, Ded1p, vasa
Chromatin remodeling	Rad54, ATRX, BLM, CHD4
Maintenance of telomeres	Pif1, Dna2, Rtel 1, WRN, BLM, FANC
Okazaki fragment maturation	Dna2, Pif1, WRN

References are cited within [19, 21, 24, 28, 55–58, 61, 63–65, 90–92, 135, 150, 159, 163, 179, 191, 198–226]

Helicases alter DNA and RNA structures, remodel chromatin [24, 51, 61–65], and modulate access to the DNA template by transcriptional machinery. RNA polymerases that are involved in the elongation of RNA transcripts have been considered as helicases that unwind the dsDNA to expose the ssDNA strand that serves as the template for RNA synthesis [2]. Helicases thus play a role in most transcriptional processes including activation (TFIIH), initiation (TFIIH, SNF2), maintenance (SW1), DNA repair (TFIIH, ERCC6/RAD26), and termination (Factor 2, Rho) [49, 66–72].

Properties of Helicases

Some of the fundamental properties exhibited by helicases are nucleic acid binding, ATP binding and hydrolysis, translocation, unwinding of duplex nucleic acids, and displacement of proteins bound to the nucleic acid substrate (Fig. 2.1), although not all helicases are able to perform all of these activities. Helicases unwind DNA with a unique directionality (either from 5′ to 3′ or from 3′ to 5′) relative to the strand of DNA that is bound by the enzyme. RecBCD exhibits bipolar enzyme activity, where RecB and RecD components of the complex unwind DNA in 3′–5′ and 5′–3′ directions, respectively [73]. Other bipolar helicases are Bacillus anthracis PcrA [74] and Sulfolobus acidocaldarius HerA helicase [75].

Fig. 2.1.

Biochemical properties of helicases: (a) ATP hydrolysis-dependent unwinding of duplex DNA by helicase (blue triangle). The movement of helicase along the DNA utilizing the energy from ATP hydrolysis separates the duplex nucleic acid into single strands. (b) An ssDNA translocase (blue triangle) moves with biased directionality along the DNA powered by ATP binding and hydrolysis. The directionality of translocation can be 3′–5′ or 5′–3′. (c) A dsDNA translocase (red hexagon) moves along dsDNA. (d) Helicases can displace proteins (magenta circles) bound to the DNA strand as a result of their biased directional movement (adapted from ref. [45])

Most helicases require a short stretch of ssDNA as a loading strand in vitro, and they show a preference for binding to ssDNA over dsDNA. Many helicases (Rep, UvrD, PcrA, Dda, and HSV-UL5) are generally considered to be ssDNA translocases, while other helicases (eIF4A, RecG, and PriA) are dsDNA translocases [76, 77]. Some helicases need a replication fork-like structure on the substrate for optimum unwinding, whereas other DNA helicases can initiate unwinding from blunt-ended duplex DNA, such as RecBCD, UvrD, Rep, and RecQ [77–79]. While the ATPase activity of helicases is low in the absence of DNA, presence of ssDNA stimulates this activity [80].

Superfamily 1 Helicases

Helicases are divided into six superfamilies (SF1–6) based on the sequence identity among the conserved helicase motifs [1, 43, 47, 81]. Superfamily 1 is one of the largest classes of helicases with members that participate in virtually all steps in DNA or RNA metabolism [82–85]. SF1 includes three families (Rep/UrvD, Pif1/RecD, and Upf1 like) [86] and can be divided into groups based on the direction of translocation on ssDNA: 3′–5′ for SF1A helicases and 5′–3′ for SF1B helicases [43]. Some of the well-characterized SF1A helicases are PcrA, Rep, and UvrD T[87–92]. Well-characterized members of SF1B include RecD and Dda [93, 94] (He et al., in press). The biological functions, polarity, and active forms of some of the SF1 helicases are listed (Table 2.2).

Table 2.2.

Superfamily 1A (3′–5′) and SF1B (5′–3′) helicases

Helicase	Source	Biological function	Active form	Polarity
PcrA	Bacillus stearothermophilus	DNA repair and rolling replication of plasmids	Monomer	3′–5′
Rep	Escherichia coli	DNA replication	Dimer	3′–5′
RecB	Escherichia coli	DNA recombination	Monomer	3′–5′
UvrD	Escherichia coli	DNA replication, recombination, and repair of UV damage and mismatched base pairs	Dimer	3′–5′
Pif1	Saccharomyces cerevisiae	Maintenance of telomeres, ribosomal DNA replication, processing of Okazaki fragments	Monomer	5′–3′
Dna2	Saccharomyces cerevisiae	Maintenance of telomeres, Okazaki fragment maturation, double strand break repair, aging	Monomer	5′–3′
Dda	Bacteriophage T4	DNA replication initiation, and recombination	Monomer	5′–3′
UL5	Herpes simplex virus	Viral DNA replication	Dimer	5′–3′
RecD2	Deinococcus radiodurans	DNA repair	Monomer	5′–3′
TraI	Escherichia coli	DNA transfer during conjugation, DNA nicking	Monomer	5′–3′

References are cited within [90–92, 135, 150, 163, 191, 198, 200–203, 205, 209, 211, 212, 214, 215, 218, 219, 225, 227–233]

Helicase Motifs

A characteristic feature of helicases is the presence of highly conserved amino acid sequences termed the “helicase motifs” [46, 47, 95–98]. Based on their sequence similarity and organization, these motifs are useful in the grouping of helicases into different families. SF1 and SF2 contain at least seven conserved amino acid motifs whose sequences, organization, and secondary structures are, in general, very similar [43, 46]. SF1 (Rep and PcrA) and SF2 (NS3) helicases differ primarily in motifs III and IV. In NS3, motif IV makes contacts with the DNA backbone and is not in the same relative position as motif IV of Rep and PcrA. While motif III of Rep contacts the bound ssDNA molecule, this motif in NS3 does not [99, 100]. The SF1A and SF1B helicases also show differences with motifs Ia and III being particularly characteristic for each class. These motifs are usually clustered in a core region of 200–700 amino acids, separated by stretches of low sequence but high length conservation [79]. In contrast, the N-terminal and C-terminal regions of helicases are characterized by a high degree of sequence and length variability. The divergent regions are responsible for individual protein functions, whereas the highly conserved motifs are involved in ATP binding and hydrolysis or binding and unwinding of nucleic acids.

Helicase motifs involved in ATP binding are located at the interface between two RecA-like domains [43, 46, 47, 95, 96, 101] in the structures of SF1 and SF2 helicases [48, 102–104]. Figure 2.2 shows the conserved sequence of the motifs (Q, I, Ia, II, III, IV, V, and VI) of the SF1 helicase family and their location in PcrA. The biochemical functions of these motifs are described below.

Fig. 2.2.

Schematic representation of the motifs of SF1 helicases: (a) The consensus sequences for the conserved helicase motifs of SF1 helicases and PcrA are shown. The N-terminus of the protein is on the left and C-terminus is on the right side. Labels below the boxes are the names assigned to the motifs (motifs Q, I, Ia, II–VI). The relative positions of motifs and spacing between motifs are arbitrary. The consensus amino acid sequences of PcrA are taken from refs. [46, 89]. (b) The crystal structure of PcrA helicase (Protein Data Bank code 3PJR) [87, 89] bound to DNA (dark green) illustrating the different conserved motifs. The helicase motifs are in the cleft formed between the two RecA-like domains (grey). The colors of different motifs in the structure are as follows: motif Q, blue; motif I, orange; motif Ia, cyan; motif II, magenta; motif III, yellow; motif IV, red; motif V, green; motif VI, purple

Q Motif

The Q of the Q motif [101] is conserved among all SF1 helicases [86]. This motif coordinates the adenine base and is less conserved among those helicase families which do not show specificity for ATP [86]. Mutagenesis suggests that the Q motif is required for viability and plays a role in orienting ATP for hydrolysis [101, 105].

Motif I (Walker A)

The consensus sequence of this motif is AxxGxGKT [46]. It is present in many nucleotide-binding proteins and forms a phosphate-binding loop [106]. The residues “GKT” are required for the interaction of the protein with Mg²⁺ and ATP [107]. The conserved G in GKT helps to maintain the flexible loop conformation [46].

Motif Ia

The consensus sequence for motif Ia is TxxAA. It has been suggested that this motif is involved in ssDNA binding [89] and energy transduction from the ATP-binding site to the DNA-binding site [86]. For the SF1 helicases (UvrD, Rep, Pif1), motifs Ia and III have been proposed to play important roles in defining translocation polarity [94].

Motif II (Walker B) [108]

This motif, DExx, is involved in NTP hydrolysis [46, 76, 86]. The D and E residues coordinate the ATP-associated Mg²⁺ and activate the attacking water molecule, respectively [109, 110]. Mutation of these residues reduces ATPase and helicase reactivity [111–113].

Motif III

The consensus sequence for motif III is GDxxQLPP [86]. It functions in DNA binding through base stacking and hydrogen bonding with the bases [76]. The close proximity of some residues in motifs III to that in motif II suggests that motif III transduces the energy of ATP hydrolysis to the DNA [88]. Motif III mutants of UL5, UvrD, and eIF-4A exhibited uncoupling of ATPase and helicase activities [79, 110, 114]. The highly conserved Q in motif III contacts the γ-phosphate of the bound nucleotide in PcrA [89], UvrD, and UL5 [113, 115]. These results imply a role for motif III in coupling ATP hydrolysis with the unwinding of duplex DNA.

Motif IV

The consensus sequence for this motif varies among the three families within SF1. Motif IV supplies a stacking platform (conserved Y) for the adenine base [88] as well as direct contact with the γ-phosphate, suggesting that it may be involved directly in hydrolysis of the NTP [89].

Motif V

This motif interacts with the sugar-phosphate backbone of the DNA. Motif V mutants of UL5 exhibited reduced affinity for ssDNA and reduced rates of ATP hydrolysis [116].

Motif VI

The consensus sequence for this motif is VA(L/Y)TRA(K/R) [86]. It is proposed to be a part of the ATP-binding cleft and is involved in coupling the helicase and ATPase activities of the protein [88]. Several helicases exhibited nucleic acid binding defects when motif VI residues were altered [110]. Motif VI mutants of UvrD exhibited reduced ssDNA binding, ATP hydrolysis rate, and ligand-induced conformational changes [117]. In motif VI mutants of UL5, an uncoupling of ATPase and helicase activity was observed [113]. Studies on PcrA [89] and Upf1p [112, 113, 117] suggest that motif VI, by virtue of its close proximity to the NTP-and DNA-binding sites, mediates ligand-induced conformational changes, which are essential for the helicase to move along the nucleic acid substrate [88].

The conserved motifs bind and hydrolyze ATP and transduce the resulting energy to cause conformational changes in the helicase. These motifs function together to drive directional movement along ssDNA or dsDNA. They participate in the communication between nucleic acid and ATP-binding sites [118]. The ability to unwind dsDNA appears to be provided by additional protein domains which do not contain the helicase motifs [48].

Helicase Structure

SF1 helicases constitute one of the best structurally characterized helicase families. The crystal structures have revealed that the helicase motifs are clustered together in two RecA-like domains, forming an ATP-binding pocket between them and a part of the nucleic acid-binding site. The nonconserved regions may contain specific domains such as protein–protein interaction domains, cellular localization domains, and DNA-recognition domains specific to individual helicases. Several helicase structures have been solved in the last decade contributing significantly to the overall understanding of the mechanism of SF1 helicases. Figure 2.3 shows the structures of two SF1A and two SF1B helicases [87–89, 93, 94, 103] (He et al., in press).

Fig. 2.3.

Crystal structures of SF1A (PcrA, UvrD) and SF1B (RecD2, Dda) helicases: (a) Ribbon diagram of PcrA helicase from Bacillus stearothermophilus (Protein Data Bank code 3PJR) [87, 89]. The RecA-like domains 1A and 2A are shown in grey and green colors, respectively. The structure shows the red 1B domain and the pin (purple) separating the strands of duplex DNA. The 2B domain is shown in cyan. (b) Structure of Escherichia coli UvrD helicase (PDB code 2IS1 [122]). Domains are colored as in (a). (c) Structure of RecD2 helicase from Deinococcus radiodurans (PDB code 3GPL [93]). Domains 1A, 2A, and 2B are colored as in (a). The beta-hairpin (1B) is red. The N-terminal domain is colored orange. (d) Structure of bacteriophage T4 Dda helicase bound to ssDNA (PDB id: 3UPU) (He et al., in press). The domains are colored as in (c). Nucleic acid is colored yellow in all structures

Structure of SF1A Helicases (PcrA and UvrD)

The first helicase structure to be solved was that of PcrA from B. stearothermophilus [87, 89]. PcrA is composed of four domains (1A, 2A, 1B, and 2B) (Fig. 2.3a), resembling other SF1 helicases [119–121]. The ATP-binding site is situated in a cleft between the RecA-like domains (1A and 2A). This cleft opens and closes in response to nucleotide binding and hydrolysis suggesting how translocation could occur [88, 89]. In UvrD (SF1A helicase), binding of an ATP analog, AMPPNP, in the cleft between domains 1A and 2A (Fig. 2.4) induces a 20° rotation between domain 2A and the remaining three domains (1A, 1B, and 2B). Upon AMPPNP binding, the duplex moves domains 1A/1B/2B towards 2A leading to untwisting of the duplex DNA as single-stranded DNA is pulled through the active site [122]. The structural data for UvrD, as for PcrA, predict one nt translocated and one bp unwound per ATP hydrolyzed.

Fig. 2.4.

Crystal structures of (a) UvrD–DNA and (b) UvrD–DNA–AMPPNP complexes [122]. The four domains (1A, 1B, 2A, and 2B) are colored grey, red, green, and cyan, respectively. The Pin region is shown in purple. The 3′-ssDNA tail is bound across domains 1A and 2A. Domains 1B and 2B interact with the DNA duplex. Binding of AMPPNP (brown) induces domains 2A and 1A to rotate towards each other by 20°

Structure of SF1B Helicases (RecD2 and Dda)

A crystal structure of Deinococcus radiodurans RecD2 helicase with ssDNA [93] is shown in Fig. 2.3c. RecD2 comprises five domains: the N-terminal, 1A, 1B, 2A, and 2B domains. Domains 1A and 2A have the RecA-like fold seen in all SF1 and SF2 helicases [48]. Domain 1B forms a rigid β-hairpin that protrudes from the surface of domain 1A, and the 2B domain has an SH3 fold [93]. The ssDNA-binding site runs in a 5′–3′ direction along a channel across the top of domains 2A and 1A. The DNA is also contacted by the 1B and 2B domains that form the sides of the channel.

Although the location of the ssDNA-binding site is similar in SF1A and SF1B helicases [88, 89], the contacts between the protein and the DNA and conformation of the DNA are different. PcrA interacts with the DNA through stacking of aromatic side chains with the DNA bases and there are relatively few contacts with the DNA backbone [89]. In contrast, the majority of protein–DNA contacts in RecD2 are via the phosphodiester backbone [93]. The bound ssDNA is in a configuration more similar to that found in a DNA duplex, with the bases stacked against one another, unlike the extended conformation observed in PcrA. Interestingly, the RecD2 mode of binding is more similar to that seen in SF2 enzymes such as NS3, Rad54, Vasa, and Hel308 [104, 123–125] rather than SF1A helicases [126].

Even though there is significant structural similarity between SF1A and SF1B enzymes, they translocate in opposite directions. Comparison of the structures of PcrA and RecD2 shows that both SF1A and SF1B helicases bind the ssDNA in the same orientation (2A domain on the 5′ side of the DNA, 1A domain on the 3′ side of the DNA) [89, 94], and reveals how directionality is determined (Fig. 2.5a, b) [89, 94, 127]. Opening and closing of the cleft between the 1A and 1B domains in the presence and absence of ATP appears to provide the means of translocation [89]. The RecA-like domains bind to DNA and upon binding to ATP, the more weakly bound domain shifts towards the more tightly bound domain. For PcrA (SF1A), domain 1A moves towards domain 2A upon ATP binding, resulting in translocation in the 3′–5′ direction [94, 127]. For RecD2 (SF1B), ATP binding causes movement of domain 2A towards domain 1A, resulting in 5′–3′ translocation [94]. The net forward movement occurs in one nt physical steps with each ATP hydrolyzed. The efficiency of helicases may vary, with some ATP hydrolysis events being uncoupled from movement. Despite being in the same superfamily, SF1A and SF1B helicases exhibit significantly different mechanisms.

Fig. 2.5.

Comparison of the translocation mechanism of SF1A (PcrA) and SF1B (RecD2) helicases: In both enzyme classes, a cycle of ATP binding and hydrolysis induces conformational changes that result in translocation of the protein along DNA, but in opposite directions. Dark grey circles represent domains that have a tight grip on the ssDNA, and light grey circles represent domains that have a weaker grip and can slide along the DNA. Conversion between tight and weak grip (dark and light grey circles) is indicated by arrows. (a) The translocation mechanism of PcrA is shown in cartoon form demonstrating the change in affinity for ssDNA of domains 1A and 2A during translocation. Prior to ATP binding, ssDNA is bound to the enzyme spanning the 1A and 2A domains. Binding of ATP induces closure of the cleft between 1A and 2A domains. At this point, the grip is tightest on the 2A domain, causing the DNA to slide across the 1A/1B domains. Upon ATP hydrolysis, bind to ssDNA in 1A becomes tighter, whereas binding of ssDNA in 2A becomes weaker, releasing ssDNA. The domains also move apart, due to domain 2A sliding forward, causing the ssDNA to be pulled along the DNA-binding channel relative to the domain 2A. The result is translocation along the DNA in a 3′–5′ direction (indicated by black arrows). (b) Translocation mechanism of RecD2 helicase. When ATP binds to the enzyme, the cleft closes between 1A and 2A motor domains, causing domains 2A and 2B to slide along the DNA backbone (black arrows). The contacts between domain 1A and the DNA remain tight to anchor the DNA as domains 2A and 2B slide along it. When the conformational change is complete, the grip of domain 1A on the DNA is loosened. Then, ATP hydrolysis takes place, allowing the cleft to relax to the open conformation. The DNA is pulled back by domains 2A and 2B, which now have a tighter grip on bound DNA than domain 1A. This causes the DNA to slide across the surface of domain 1A as it moves away from domains 2A and 2B. The result is translocation by one base in a 5′–3′ direction (black arrows) during a single round of ATP binding and hydrolysis (adapted from ref. [94])

Duplex DNA Is Split by the Pin Region

In addition to the RecA-like 1A and 2A domains containing the conserved helicase motifs, all SF1 helicases contain accessory domains which can vary in structure. One common feature of these domains is the presence of a pin or wedge which functions to split the incoming DNA. The pin was first discovered in the SF2 helicase Hel308 from Archaeoglobus fulgidus [125]. A pin splitting the DNA was observed previously in RecC [103], the Chi recognition protein of the RecBCD helicase complex and in RuvA [128, 129], which along with RuvB and RuvC catalyzes branch migration resulting in Holliday junction resolution.

The location of the pin varies to correspond with the direction of translocation. For SF1A helicases, the 2A domain leads during unwinding in the 3′–5′ direction. In the structures of PcrA with DNA bound (Fig. 2.3a) [89] a pin is visible, positioned at the junction of the duplex and ssDNA. For SF1B helicases, 1A is the leading RecA-like domain, and an insertion in this domain, domain 1B, serves as the pin (Fig. 2.3c, d) [93]. In each case, the pin is positioned appropriately to split the incoming duplex during translocation along the ssDNA. Based on the structure of RecD2 bound to DNA, a mutant lacking the pin was designed which was completely devoid of unwinding activity although it hydrolyzed ATP at the same rate as wt RecD2 [93].

A recent report demonstrates that the mere presence of the pin is not sufficient for helicase activity (He et al., in press). Specific residues in the pin may be necessary for helicase activity. For the SF1B helicase Dda, mutation of a single F residue in the pin which stacks with the ssDNA completely eliminates strand separation although translocation on ssDNA is unaffected. In the case of RecD2 [93], the pin is short and it appears to function only in splitting the incoming duplex. Dda contains a long β-hairpin which is anchored at its tip by electrostatic interactions to domain 2B, allowing it to function not only directly in splitting the dsDNA but also in coupling ATP hydrolysis to unwinding (He et al., in press). Like Dda, the hepatitis C virus NS3 helicase (SF2) contains an extended pin [100], and mutation of conserved residues with this pin also uncouples ATP hydrolysis from unwinding [130].

Ensemble Kinetics for Helicase-Catalyzed DNA Unwinding of dsDNA and Translocation on ssDNA

SF1 helicases unwind the DNA in a stepwise manner so that more steps are required to unwind longer duplexes. Unwinding of duplexes of varying length has led to several descriptors of the kinetic and physical constants associated with helicases. One of the most discussed values relates to the “step size.” The kinetic step size refers to the number of base pairs unwound prior to a rate-limiting kinetic step. This value can be determined by measuring unwinding of increasing length duplexes [131]. The kinetic step sizes for some SF1 helicases are shown in Table 2.3. The physical step size refers to the number of base pairs that are unwound simultaneously. Single-molecule approaches have provided direct measures of the physical step size for a number of helicases (see below). The relationship between the kinetic step and the physical step size may be complex. A helicase might unwind one base pair at a time (physical step of one), but then proceed through a slow conformational change that occurs every three base pairs, resulting in a kinetic step size of three bps. The chemical step size refers to the number of base pairs unwound per ATP hydrolyzed. In the simplest case, all of these values are equal to one. However, there may be differences and care must be taken to distinguish between these values when comparing the activity of one helicase to another.

Table 2.3.

Kinetic constants for select SF1 helicases

Helicase	Kinetic step size for unwind (bp)	Kinetic step size for trans (nt)	Unwind rate (bp/s)	Trans. rate (nt/s)	Unwinding processivity (bp)	Trans. processivity (nt)
wtRep—monomer	N.D.a	N.D.	N.D.	298 [155]	0 [119]	700 [155]
wtRep—EEb	4c [119]	N.D.	57 [119]	N.D.	N.D.	N.D.
ReΔ2B—monomer	4c [155]	N.D.	226 [155]	530 [155]	<21 [155]	800 [155]
UvrD—monomer	N.D.	4–5 [92, 156, 234]	N.D.	200 [92, 156, 234]	<18 bp [156]	240 [92, 234]
UvrD—EE	4.4 [131]	N.D.	73.4 [131]	N.D.	10 [131]	N.D.
RecBCD EE	3–4 [235–238]	N.D.d	700–1,350 [235–239]	N.D.d	30,000 [183]	N.D.
PcrA—monomer	N.D.	4.0 [145]	N.D.	234 [145]	<18 [145]	248 [145]
PcrA—EE	4 [240]	N.D.	31 [145, 240]	N.D.	N.D.	N.D.
Dda—monomer	2.8 [241, 242]	N.D.	262 [136, 241, 242]	267 [136]	19 [242]	N.D.
Dda—EE	3.4 [242]	N.D.	257 [242]	N.D.	64 [242]	N.D.
TraI—monomer	6.2 [159]	N.D.	1,085 [159]	N.D.	N.D.	N.D.
TraI EE	8.2 [159]	N.D.	1,120 [159]	N.D.	>850 [243]	N.D.

^aNot determined

^bExcess enzyme—enzyme concentration is in excess of substrate

^cStep size for ReΔ2B is assumed based on the duplex length and the number of steps in the kinetic mechanism

^dKinetic step size and rates of translocation have been measured for RecB and RecBC, but not for RecBCD [244]

Similar to the unwinding studies, translocation can be examined by measuring the time needed to reach the end of varying lengths of ssDNA. The lag phase for these measurements provides kinetic information that can give rise to kinetic step sizes, rates of translocation, and coupling efficiencies when combined with ATP hydrolysis measurements. One assay that provided a breakthrough in helicase ATPase studies was the development of the phosphate-binding protein for measuring phosphate release kinetics [91]. This assay has been instrumental in relating the rates and efficiencies of ATPase activity to movement on ssDNA. However, it has not been generally applied, as yet, to understanding unwinding of dsDNA.

Helicases can be described as acting by an active or passive mechanism in reference to whether they actively separate the duplex or simply trap single-stranded intermediates that form as a result of thermal fraying [132, 133]. One suggestion for classifying active vs. passive helicases relies on comparing the ratio of the velocity for translocation on ssDNA to the velocity for unwinding of dsDNA [133]. If this ratio falls between 0.25 and 1, then a helicase can be considered as active. Most helicases likely fall between these extremes, and for some SF1 helicases, comparison of unwinding and translocation rates is complicated by oligomerization that occurs during unwinding [119, 134, 135]. However, bacteriophage T4 Dda has been shown to unwind duplex DNA and translocate on ssDNA at the same rate suggesting that it functions by a completely active mechanism [136].

Single-Molecule Methods Provide New Insights into SF1 Helicases

Breakthroughs in technology have resulted in corresponding breakthroughs in biology, and this theme has held true in understanding helicase mechanisms. Single-molecule Förster resonance energy transfer (smFRET) as well as laser tweezers or magnetic tweezers have been extensively applied to the study of helicases during the past decade [137, 138]. These techniques are particularly useful for visualizing kinetic events that are “hidden” within ensemble experiments. Recognition of the Chi sequence in DNA causes RecBCD to pause and reduce its translocation rate to approximately one-half the initial rate [139–141] resulting in a switch in motor usage with RecD being the lead motor prior to Chi and RecB after the Chi sites [142]. Magnetic tweezer analysis reported the average unwinding rate by RecBCD to be 900 bp/s [143] and the processivity of the complex to be ~1.

The physical step size can be directly observed in some cases using smFRET or laser tweezers. Single-molecule studies of PcrA reported the unwinding step size to be one nt [144], whereas a kinetic step size of four nts for translocation was estimated from ensemble experiments [145]. The larger kinetic step size determined from ensemble analysis could be an overestimation due to the presence of static disorder. In the case of Dda, single-molecule and ensemble experiments reported the unwinding rate to be ~250 bp/s and the rate varied little for forces ranging from 5 to 13 pN [136].

The mechanisms by which helicases catalyze protein displacement are beginning to be explored [146]. Single-molecule studies revealed the repetitive movements of Rep, PcrA, and UvrD helicases on the same stretch of DNA. The in vitro smFRET studies showed that the shuttling of the Rep monomer on ssDNA can prevent RecA filament formation [147], and that PcrA reeling in ssDNA can remove a preformed RecA filament [144]. Two other SF1 helicases, yeast Srs2 [148, 149] and UvrD [150], can displace Rad51 and RecA presynaptic filaments from ssDNA, respectively. Single-molecule studies offer insight into why many helicases display only limited unwinding processivity in vitro. For Rep, the reduced processivity in vitro is due to the relative instability of the functional complex [120]. The open and closed conformational states of Rep helicase undergoing ATP hydrolysis while bound to DNA were studied using smFRET [151]. The biological significance of having multiple conformations might be to regulate the helicase activity. Recent developments in three (or more)-color FRET [152, 153] should enable one to obtain simultaneous information on more than one activity, for example, ATP cycling and movement on DNA.

Protein–Protein Interactions That Regulate Helicase Activity

Helicases translocate along nucleic acids while separating dsDNA into single strands. Translocase activity alone is, in some cases, insufficient for helicase activity. In these cases, oligomerization and/or interactions with other proteins can regulate their translocase and helicase activities. Oligomerization can affect their NTPase, DNA-binding and -unwinding activities [45]. The monomeric forms of some SF1 enzymes, Rep [119, 120, 147, 154, 155], UvrD [121, 134, 156], and PcrA [89, 91, 127, 145], are processive translocases [91, 92, 156, 157], but do not display DNA unwinding activity in vitro [147]. On the other hand, the SF1 helicases Dda [158] and TraI [159] are able to function as monomeric helicases in vitro, b. But with the exception of the TraI, the SF1 helicases, when examined by themselves, generally unwind DNA with low processivity in vitro.

The nucleic acid unwinding processivity of some SF1 helicases can be increased significantly either through self-assembly or interactions with accessory proteins [119, 121, 134, 135, 160]. Rep helicase (SF1A) exists as a monomer in the absence of DNA [161]. However, Rep undergoes a DNA-induced dimerization upon binding either ss or dsDNA [132, 162], and the dimer appears to be the active form of the Rep helicase [119, 132, 161, 163–165]. Single turnover kinetic studies of UvrD-catalyzed DNA unwinding suggested that dimers are the minimal oligomeric form needed for optimal helicase activity [121, 134, 135]. In the case of Pif1, binding of ssDNA induces protein dimerization [166]. Oligomerization provides the active helicase with multiple DNA- and NTP-binding sites that are necessary for optimal DNA unwinding activity.

Regulation of helicase activity through protein–protein interactions may occur by altering conformations of the helicase. Crystal structures of Rep bound to ssDNA showed open and closed forms that differ in the 2B domain orientation [88] (Fig. 2.6). Deletion of the 2B domain in Rep was found to activate the helicase activity [154, 155]. Although, earlier smFRET studies have suggested the closed form of Rep to be an inhibited form [167], later detailed studies revealed two distinct Rep-partial-duplex DNA conformations in the ATPγS and ADP states. Here the primary conformation is found to be similar to the closed form, and in the secondary conformation the duplex DNA and 2B domain are rotated relative to the rest of the protein [151]. The multiple conformations may provide a mechanism of regulation of helicase activity whereby interactions between Rep and other proteins may determine the relative conformational states of domain 2B.

Fig. 2.6.

Crystal structure of E. coli Rep helicase (PDB code 1UAA [88]) in the open and closed conformations. Rep consists of four domains 1A, 1B, 2A, and 2B which are colored grey, red, green, and cyan, respectively. The open (a) and closed (b) conformations differ by rotations of around 130° of the 2B domain about a hinge region connecting it to the 2A domain [167]. The other domains are unchanged in both forms

In addition to helicases interacting among themselves, they can interact with other helicases to modulate their activity. It has been reported that Pif1 helicase activity is essential in top3 mutants in an Sgs1-dependent manner [168]. Pif1 has been suggested to strip Sgs1 from DNA, thereby downregulating the activity of Sgs1. Srs2 helicase has been reported to have a similar role in the disassembly of Rad51 filaments [148, 149]. Interaction between Rep and DnaB has been suggested to promote fork progression along protein-bound DNA [169].

Many SF1 enzymes are poor helicases in vitro; therefore, it is not surprising that their activities are enhanced through interactions with accessory proteins. Several helicases from prokaryotes and eukaryotes interact with other proteins to stimulate helicase activity [170–174]. The phage Φx174 gene A protein increases the helicase processivity of Rep [175–178]. PcrA is a nonprocessive helicase with difficulty unwinding even short lengths of duplex DNA [145], but the presence of plasmid replication initiator protein, RepD, enables PcrA to separate duplexes with high processivity [160, 179, 180].

The RecBCD holoenzyme contains two motors RecB (SF1A) and RecD (SF1B) in addition to the Chi recognition protein RecC [142]. RecBCD is compared to the world’s fastest supercar [181], since with the RecB and RecD helicase motors [73, 182], RecBCD is capable of moving along DNA at over 1,000 base pairs per second [183]. RecBCD is able to switch which of the two motors takes the lead, thereby regulating the translocation velocity of the complex [142] following the recognition of recombination hotspots called Chi sites [184, 185]. RecB helicase is activated through an interaction with RecC. RecB is a poor helicase by itself [186], but in complex with RecC is highly processive [139, 182, 183, 187]. Interaction with the accessory protein RecC is suggested to relieve an inhibitory function of the 2B subdomain of RecB [188]. Similarly, the inability of Rep monomers to function as helicases in vitro seems to be the result of an autoinhibitory effect of its subdomain 2B, and the deletion of this domain stimulates helicase activity of the Rep monomer [155].

Increased helicase processivity has been linked to helicase–SSB interactions [173, 189]. Rep and UvrD advance movement of replisomes blocked by nucleoprotein complexes in vitro [190]. Here the binding of successive monomers of Rep or UvrD at a blocked fork could facilitate protein displacement. Okazaki fragment processing in eukaryotes can occur either by the FEN1-only pathway or the two-nuclease pathway [191]. It has been reported that Dna2, Pif1, and RPA, the proteins of the two-nuclease pathway, stimulate FEN1 acting in the one-nuclease pathway [192]. Interactions with these proteins may change the conformation of FEN1 to optimally function on the substrate [192].

Saccharomyces Rrm3 helicase (SF1B) promotes replication fork progression through telomeric and subtelomeric DNA. Rrm3 is telomere-associated in vivo, suggesting a direct role in telomere replication [193]. Rrm3 is also needed for the timely replication of the entire genome, possibly through its role in promoting fork progression through difficult-to-replicate sites [194]. Rrm3 interacts with the catalytic subunit of DNA polymerase epsilon as it moves through both Rrm3p-dependent and -independent sites [194].

In addition to the stimulatory effects, some helicase-accessory protein interactions reduce the helicase activity. Through structural, biochemical, and functional studies, it was shown that the Srs2 helicase interacts with SUMO-PCNA thereby suppressing the Rad52-dependent recombinational repair pathway [195]. Also, Pif1 helicase negatively regulates telomere lengths by catalytically inhibiting telomerase activity [196–198].

Conclusion

The overall view from these studies is that SF1 helicases have multiple DNA-binding sites and that the entire enzyme does not move as a single unit, but instead different domains of the enzyme move at different times during the translocation–unwinding cycle as a function of ATP binding and hydrolysis. The description of this movement as an “inchworm” appears to hold true. Although the structures and helicase motifs are quite similar, the in vivo activities vary, which are likely achieved through structural differences outside of the 1A and 2A (RecA-like) domains. These variations can affect how the helicase interacts with the DNA substrate and how it interacts with other proteins which regulate its activity.

Despite the impressive progress made in understanding the kinetic and chemical mechanisms of helicases, there is much to be learned. The specific mechanism(s) by which most helicases actively pry apart the duplex remain to be determined. The specific step in the overall mechanism that limits the rate of the reaction is generally not known. Some data suggest that steps in the ATP hydrolysis cycle such as release of phosphate may limit the overall rate of DNA unwinding. The interactions between protein and DNA with the loading strand (or translocase strand) have been defined but interactions with the displaced strand largely remain a mystery. The role of protein–protein interactions in helicase mechanisms and other regulatory mechanisms remains to be uncovered. Finally, the specific mechanisms whereby helicases displace other proteins from DNA remain to be determined. There are certainly many unanswered questions that will require continued growth in the field.