Eric J. Tomko

Non-hexameric DNA helicases and translocases: mechanisms and regulation.

Helicases and nucleic acid translocases are motor proteins that have essential roles in nearly all aspects of nucleic acid metabolism, ranging from DNA replication to chromatin remodelling. Fuelled by the binding and hydrolysis of nucleoside triphosphates, helicases move along nucleic acid filaments and separate double-stranded DNA into their complementary single strands. Recent evidence indicates that the ability to simply translocate along single-stranded DNA is, in many cases, insufficient for helicase activity. For some of these enzymes, self assembly and/or interactions with accessory proteins seem to regulate their translocase and helicase activities.

Srs2 Disassembles Rad51 Filaments by a Protein-Protein Interaction Triggering ATP Turnover and Dissociation of Rad51 from DNA

Rad51 is a DNA recombinase functioning in the repair of DNA double-strand breaks and the generation of genetic diversity by homologous recombination (HR). In the presence of ATP, Rad51 self-assembles into an extended polymer on single-stranded DNA to catalyze strand exchange. Inappropriate HR causes genomic instability, and it is normally prevented by remodeling enzymes that antagonize the activities of Rad51 nucleoprotein filaments. In yeast, the Srs2 helicase/translocase suppresses HR by clearing Rad51 polymers from single-stranded DNA. We have examined the mechanism of disassembly of Rad51 nucleoprotein filaments by Srs2 and find that a physical interaction between Rad51 and the C-terminal region of Srs2 triggers ATP hydrolysis within the Rad51 filament, causing Rad51 to dissociate from DNA. This allosteric mechanism explains the biological specialization of Srs2 as a DNA motor protein that antagonizes HR.

Bacillus stearothermophilus PcrA Monomer Is a Single-stranded DNA Translocase but Not a Processive Helicase in Vitro

Structural studies of the Bacillus stearothermophilus PcrA protein along with biochemical studies of the single-stranded (ss) DNA translocation activity of PcrA monomers have led to the suggestion that a PcrA monomer possesses processive helicase activity in vitro. Yet definitive studies testing whether the PcrA monomer possesses processive helicase activity have not been performed. Here we show, using single turnover kinetic methods, that monomers of PcrA are able to translocate along ssDNA, in the 3′ to 5′ direction, rapidly and processively, whereas these same monomers display no detectable helicase activity under the same solution conditions in vitro. The PcrA monomer ssDNA translocation activity, although necessary, is not sufficient for processive helicase activity, and thus the translocase and helicase activities of PcrA are separable. These results also suggest that the helicase activity of PcrA needs to be activated either by self-assembly or through interactions with accessory proteins. This same behavior is displayed by both the Escherichia coli Rep and UvrD monomers. Hence, all three of these SF1 enzymes are ssDNA translocases as monomers but do not display processive helicase activity in vitro unless activated. The fact that the translocase and helicase activities are separable suggests that each activity may be used for different functions in vivo.

5′‐Single‐stranded/duplex DNA junctions are loading sites for E. coli UvrD translocase

Escherichia coli UvrD is a 3′–5′ superfamily 1A helicase/translocase involved in a variety of DNA metabolic processes. UvrD can function either as a helicase or only as an single‐stranded DNA (ssDNA) translocase. The switch between these activities is controlled in vitro by the UvrD oligomeric state; a monomer has ssDNA translocase activity, whereas at least a dimer is needed for helicase activity. Although a 3′‐ssDNA partial duplex provides a high‐affinity site for a UvrD monomer, here we show that a monomer also binds with specificity to DNA junctions possessing a 5′‐ssDNA flanking region and can initiate translocation from this site. Thus, a 5′‐ss–duplex DNA junction can serve as a high‐affinity loading site for the monomeric UvrD translocase, whereas a 3′‐ss–duplex DNA junction inhibits both translocase and helicase activity of the UvrD monomer. Furthermore, the 2B subdomain of UvrD is important for this junction specificity. This highlights a separation of helicase and translocase function for UvrD and suggests that a monomeric UvrD translocase can be loaded at a 5′‐ssDNA junction when translocation activity alone is needed.

Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation

Significance How ATP hydrolysis is coupled to promoter DNA unwinding and open complex formation at RNA polymerase II (Poll II) promoters is a longstanding question. Of the multisubunit RNA polymerases, only Pol II requires ATP for DNA unwinding. Here we show that the general transcription factor TFIIH subunit Ssl2 is a double-stranded DNA translocase. These and other data suggest that Ssl2 promotes DNA opening by tracking along the nontemplate promoter strand, rotating and inserting DNA into the Pol II active site cleft, leading to DNA unwinding. Our accompanying biochemical studies explain why the open complex is unstable and how TFIIH can promote Pol II escape from the promoter. Our findings also have important implications for the mechanism of TFIIH-mediated DNA repair. Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5′ → 3′ direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

Translocation of Saccharomyces cerevisiae Pif1 helicase monomers on single-stranded DNA

In Saccharomyces cerevisiae Pif1 participates in a wide variety of DNA metabolic pathways both in the nucleus and in mitochondria. The ability of Pif1 to hydrolyse ATP and catalyse unwinding of duplex nucleic acid is proposed to be at the core of its functions. We recently showed that upon binding to DNA Pif1 dimerizes and we proposed that a dimer of Pif1 might be the species poised to catalysed DNA unwinding. In this work we show that monomers of Pif1 are able to translocate on single-stranded DNA with 5′ to 3′ directionality. We provide evidence that the translocation activity of Pif1 could be used in activities other than unwinding, possibly to displace proteins from ssDNA. Moreover, we show that monomers of Pif1 retain some unwinding activity although a dimer is clearly a better helicase, suggesting that regulation of the oligomeric state of Pif1 could play a role in its functioning as a helicase or a translocase. Finally, although we show that Pif1 can translocate on ssDNA, the translocation profiles suggest the presence on ssDNA of two populations of Pif1, both able to translocate with 5′ to 3′ directionality.

Active displacement of RecA filaments by UvrD translocase activity

The UvrD helicase has been implicated in the disassembly of RecA nucleoprotein filaments in vivo and in vitro. We demonstrate that UvrD utilizes an active mechanism to remove RecA from the DNA. Efficient RecA removal depends on the availability of DNA binding sites for UvrD and/or the accessibility of the RecA filament ends. The removal of RecA from DNA also requires ATP hydrolysis by the UvrD helicase but not by RecA protein. The RecA-removal activity of UvrD is slowed by RecA variants with enhanced DNA-binding properties. The ATPase rate of UvrD during RecA removal is much slower than the ATPase activity of UvrD when it is functioning either as a translocase or a helicase on DNA in the absence of RecA. Thus, in this context UvrD may operate in a specialized disassembly mode.

Force-dependent melting of supercoiled DNA at thermophilic temperatures

Local DNA opening plays an important role in DNA metabolism as the double-helix must be melted before the information contained within may be accessed. Cells finely tune the torsional state of their genomes to strike a balance between stability and accessibility. For example, while mesophilic life forms maintain negatively superhelical genomes, thermophilic life forms use unique mechanisms to maintain relaxed or even positively supercoiled genomes. Here, we use a single-molecule magnetic tweezers approach to quantify the force-dependent equilibrium between DNA melting and supercoiling at high temperatures populated by Thermophiles. We show that negatively supercoiled DNA denatures at 0.5 pN lower tension at thermophilic vs. mesophilic temperatures. This work demonstrates the ability to monitor DNA supercoiling at high temperature and opens the possibility to perform magnetic tweezers assays on thermophilic systems. The data allow for an estimation of the relative energies of base-pairing and DNA bending as a function of temperature and support speculation as to different general mechanisms of DNA opening in different environments. Lastly, our results imply that average in vivo DNA tensions range between 0.3 and 1.1 pN.

TFIIH generates a six-base-pair open complex during RNAP II transcription initiation and start-site scanning

Eukaryotic mRNA transcription initiation is directed by the formation of the megadalton-sized preinitiation complex (PIC). After PIC formation, double-stranded DNA (dsDNA) is unwound to form a single-stranded DNA bubble, and the template strand is loaded into the polymerase active site. DNA opening is catalyzed by Ssl2 (XPB), the dsDNA translocase subunit of the basal transcription factor TFIIH. In yeast, transcription initiation proceeds through a scanning phase during which downstream DNA is searched for optimal start sites. Here, to test models for initial DNA opening and start-site scanning, we measure the DNA-bubble sizes generated by Saccharomyces cerevisiae PICs in real time using single-molecule magnetic tweezers. We show that ATP hydrolysis by Ssl2 opens a 6-base-pair (bp) bubble that grows to 13 bp in the presence of NTPs. These observations support a two-step model wherein ATP-dependent Ssl2 translocation leads to a 6-bp open complex that RNA polymerase II expands via NTP-dependent RNA transcription.

Conformational selection and induced fit as a useful framework for molecular motor mechanisms

The linkage between macromolecular binding and conformational change that is ubiquitous in biological molecules can be understood in the context of the mechanisms of conformational selection and induced fit. Here, we explore mappings between these mechanisms of ligand binding and those underlying the translocation of molecular motors and the nucleic acid unwinding of helicases. The mechanism of biased motion exhibited by molecular motors is typically described as either a thermal ratchet or a power-stroke and nucleic acid helicases are characterized by either active or passive unwinding mechanisms. We posit that both Brownian ratchet translocation and passive unwinding are examples of conformational selection and that both power-stroke translocation and active unwinding are examples of induced fit. Furthermore, in ligand-binding reactions, both conformational selection and induced fit may exist in parallel leading to a ligand-dependent flux through the different mechanistic pathways. Given the mappings we describe, we propose that motors may be able to function via parallel ratchet and stroke mechanisms and that helicases may be able to function via parallel active and passive mechanisms.