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.

E. coli UvrD is an SF1 helicase involved in several DNA metabolic processes. Although a UvrD dimer is needed for helicase activity, a monomer can translocate with 3' to 5' directionality along single-stranded DNA, and this ATP-dependent translocation is likely involved in RecA displacement. In order to understand how the monomeric translocase functions, we have combined fluorescence stopped-flow kinetic methods with recently developed analysis methods to determine the kinetic mechanism, including ATP coupling stoichiometry, for UvrD monomer translocation along ssDNA. Our results suggest that the macroscopic rate of UvrD monomer translocation is not limited by each ATPase cycle but rather by a slow step (pause) in each translocation cycle that occurs after four to five rapid 1 nt translocation steps, with each rapid step coupled to hydrolysis of one ATP. These results suggest a nonuniform stepping mechanism that differs from either a Brownian motor or previous structure-based inchworm mechanisms.

SUMMARY 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.

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.DNA helicases are nucleic acid motor proteins that use nucleoside triphosphate binding and hydrolysis to unwind duplex DNA (1-4). These enzymes generally also have the ability to translocate with biased directionality along singlestranded (ss) 3 DNA (5-9). In fact, some of the biological activities of these enzymes, such as displacement of other proteins from ssDNA (10 -16), may be dependent only on the ability of these enzymes to translocate along ssDNA rather than on helicase activity. DNA helicases have been grouped into families and superfamilies defined by conserved amino acid sequence motifs (17). These enzymes can also be divided into two distinct structural classes, with one class functioning as toroidal or ringlike hexamers (4). By far, the largest number of helicases and putative helicases belong to the nonhexameric SF1 or SF2 superfamilies (17).SF1 enzymes have often been considered to be monomeric helicases (18,19). Yet direct experimental tests of whether a monomeric protein has helicase activity have been performed for only a few enzymes. The most direct experimental approach to address the question of what oligomeric form of the enzyme is needed for helicase activity requires examination of the DNA unwinding kinetics under single turnover conditions, such that only a single round of DNA binding is allowed. In such an experiment, the DNA substrate is prebound with enzyme under conditions such that the oligomeric form of the enzyme is known (e.g. a monomer), and the reaction is started by the addition of nucleoside triphosphate (e.g. ATP) along with a "trap" for free enzyme (20,21). Any DNA unwinding that is observed in such a single turnover e...

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. 1, 2). The OC forms when Pol and its associated transcription machinery bind to promoter DNA, generating a series of conformational changes in both DNA and protein, including the unwinding of ∼11 bp of DNA upstream from the transcription start site (TSS). This open state is stabilized by interactions of Pol with the unwound strands of promoter DNA and by the binding of downstream double-stranded promoter DNA to the Pol Cleft/Jaw domains (3-5). All tested multisubunit Pols spontaneously form OCs, except for RNA Pol II, where ATP and the general transcription factor TFIIH are required for DNA unwinding (6-8). For Pol II, this unwound state is unstable, decaying with a half-life of 30-60 s (8, 9).The general transcription factor TFIIH contains two subunits with DNA-dependent ATPase activity, XPD/Rad3 and XPB/Ssl2 (human/yeast proteins), which are members of the SF2 helicasetranslocase family (10). The XPB/Ssl2 ATPase is required for DNA unwinding in the OC, whereas the XPD/Rad3 ATPase activity does not function in transcription (11)(12)(13)(14). In DNA strand displacement assays, the two isolated human subunits have DNA helicase activity of opposite polarity with XPD having 5′ → 3′ activity and XPB having 3′ → 5′ activity. XPD is at least eightfold more active than XPB in strand displacement (15). In contrast to the activity of purified XPB, the most purified preparations of native or recombinant human TFIIH have only the XPD 5′ → 3′ helicase activity, with no detectable 3′ → 5′ helicase function (14,15). In addition to its role in transcription initiation, TFIIH can assist in Pol II promoter escape in an XPB-dependent mechanism (16, 17), and both the XPB and XPD subunits of TFIIH play an essential role in general and transcription-coupled nucleotide excision repair (NER) (18,19).Mapping the location of XPB/Ssl2 in RNA Pol II preinitiation complexes (PICs) revealed that this factor binds promoter DNA downstream from the site of DNA unwinding in the OC (2...