Steven W. Matson

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.

The Escherichia coli MutL Protein Physically Interacts with MutH and Stimulates the MutH-associated Endonuclease Activity

All possible pairwise combinations of UvrD, MutL, MutS, and MutH were tested using the yeast two-hybrid system to identify potential interactions involving mismatch repair proteins. A two-hybrid screen previously identified a physical interaction between MutL and UvrD. Although several other known interactions were not observed, a novel interaction between MutL and MutH was detected. A series of truncations from the NH2 and COOH termini of MutL demonstrated that the COOH-terminal 218 amino acids were sufficient for the two-hybrid interaction with MutH. Removal of a small number of residues from either the NH2 or COOH termini of MutH eliminated the two-hybrid interaction with MutL. Protein affinity chromatography experiments confirmed that MutL, but not MutS, physically associates with MutH. Furthermore, MutL greatly stimulated the d(GATC)-specific endonuclease activity of MutH in the absence of MutS and a mispaired base. Stimulation of the MutH-associated endonuclease activity by MutL was dependent on ATP binding but not ATP hydrolysis. Further stimulation of this reaction by MutS required the presence of a DNA mismatch and a hydrolyzable form of ATP. These results suggest that MutL activates the MutH-associated endonuclease activity through a physical interaction during methyl-directed mismatch repair in Escherichia coli.

Evidence for a physical interaction between the Escherichia coli methyl-directed mismatch repair proteins MutL and UvrD

UvrD (DNA helicase II) is an essential component of two major DNA repair pathways in Escherichia coli: methyl‐directed mismatch repair and UvrABC‐mediated nucleotide excision repair. In addition, it has an undefined role in the RecF recombination pathway and possibly in replication. In an effort to better understand the role of UvrD in these various aspects of DNA metabolism, a yeast two‐hybrid screen was used to search for interacting protein partners. Screening of an E.coli genomic library revealed a potential interaction between UvrD and MutL, a component of the methyl‐directed mismatch repair pathway. The interaction was confirmed by affinity chromatography using purified proteins. Deletion analysis demonstrated that the C‐terminal 218 amino acids (residues 398–615) of MutL were sufficient to produce the two‐hybrid interaction with UvrD. On the other hand, both the N‐ and C‐termini of UvrD were required for interaction with MutL. The implications of this interaction for the mismatch repair mechanism are discussed.

Nicking by transesterification: the reaction catalysed by a relaxase

DNA relaxases play an essential role in the initiation and termination of conjugative DNA transfer. Purification and characterization of relaxases from several plasmids has revealed the reaction mechanism: relaxases nick duplex DNA in a site‐ and strand‐specific manner by catalysing a transesterification. The product of the reaction is a nicked double‐stranded DNA molecule with a sequestered 3′‐OH and the relaxase covalently bound to the 5′ end of the cleaved strand via a phosphotyrosyl linkage. The relaxase‐catalysed transesterification is isoenergetic and reversible; a second transesterification ligates the nicked DNA. However, the covalent nucleoprotein complex is relatively long‐lived, a property that is likely to be essential for its role as an intermediate in the process of conjugative DNA transfer. Subsequent unwinding of the nicked DNA intermediate is required to produce the single strand of DNA transferred to the recipient cell. This reaction is catalysed by a DNA helicase, an activity intrinsic to the relaxase protein in some, but not all, plasmid systems. The first relaxase‐catalysed transesterification is essential for initiation of conjugative strand transfer, whereas the second is presumably required for termination of the process. The relaxase, in conjunction with several auxiliary proteins, forms the relaxation complex or relaxosome first described nearly 30 years ago as being associated with conjugative and mobilizable plasmids.

The UvrD helicase and its modulation by the mismatch repair protein MutL

UvrD is a superfamily I DNA helicase with well documented roles in excision repair and methyl-directed mismatch repair (MMR) in addition to poorly understood roles in replication and recombination. The MutL protein is a homodimeric DNA-stimulated ATPase that plays a central role in MMR in Escherichia coli. This protein has been characterized as the master regulator of mismatch repair since it interacts with and modulates the activity of several other proteins involved in the mismatch repair pathway including MutS, MutH and UvrD. Here we present a brief summary of recent studies directed toward arriving at a better understanding of the interaction between MutL and UvrD, and the impact of this interaction on the activity of UvrD and its role in mismatch repair.

Escherichia coli DNA Helicase II Is Active as a Monomer

Helicases are thought to function as oligomers (generally dimers or hexamers). Here we demonstrate that althoughEscherichia coli DNA helicase II (UvrD) is capable of dimerization as evidenced by a positive interaction in the yeast two-hybrid system, gel filtration chromatography, and equilibrium sedimentation ultracentrifugation (K d = 3.4 μm), the protein is active in vivo andin vitro as a monomer. A mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) failed to dimerize and yet was as active as the wild-type protein in ATP hydrolysis and helicase assays. In addition, the uvrDΔ40C allele fully complemented the loss of helicase II in both methyl-directed mismatch repair and excision repair of pyrimidine dimers. Biochemical inhibition experiments using wild-type UvrD and inactive UvrD point mutants provided further evidence for a functional monomer. This investigation provides the first direct demonstration of an active monomeric helicase, and a model for DNA unwinding by a monomer is presented.

Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase

Conjugative transfer of plasmid DNA via close cell–cell junctions is the main route by which antibiotic resistance genes spread between bacterial strains. Relaxases are essential for conjugative transfer and act by cleaving DNA strands and forming covalent phosphotyrosine linkages. Based on data indicating that multityrosine relaxase enzymes can accommodate two phosphotyrosine intermediates within their divalent metal-containing active sites, we hypothesized that bisphosphonates would inhibit relaxase activity and conjugative DNA transfer. We identified bisphosphonates that are nanomolar inhibitors of the F plasmid conjugative relaxase in vitro. Furthermore, we used cell-based assays to demonstrate that these compounds are highly effective at preventing DNA transfer and at selectively killing cells harboring conjugative plasmids. Two potent inhibitors, clodronate and etidronate, are already clinically approved to treat bone loss. Thus, the inhibition of conjugative relaxases is a potentially novel antimicrobial approach, one that selectively targets bacteria capable of transferring antibiotic resistance and generating multidrug resistant strains.

DNA helicases of Escherichia coli.

A great deal has been learned in the last 15 years with regard to how helicase enzymes participate in DNA metabolism and how they interact with their DNA substrates. However, many questions remain unanswered. Of critical importance is an understanding of how NTP hydrolysis and hydrogen-bond disruption are coupled. Several models exist and are being tested; none has been proven. In addition, an understanding of how a helicase disrupts the hydrogen bonds holding duplex DNA together is lacking. Recently, helicase enzymes that unwind duplex RNA and DNA.RNA hybrids have been described. In some cases, these are old enzymes with new activities. In other cases, these are new enzymes only recently discovered. The significance of these reactions in the cell remains to be clarified. However, with the availability of significant amounts of these enzymes in a highly purified state, and mutant alleles in most of the genes encoding them, the answers to these questions should be forthcoming. The variety of helicases found in E. coli, and the myriad processes these enzymes are involved in, were perhaps unexpected. It seems likely that an equally large number of helicases will be discovered in eukaryotic cells. In fact, several helicases have been identified and purified from eukaryotic sources ranging from viruses to mouse cells (4-13, 227-234). Many of these helicases have been suggested to have roles in DNA replication, although this remains to be shown conclusively. Helicases with roles in DNA repair, recombination, and other aspects of DNA metabolism are likely to be forthcoming as we learn more about these processes in eukaryotic cells.

DNA Unwinding by Escherichia coli DNA Helicase I (TraI) Provides Evidence for a Processive Monomeric Molecular Motor

The F plasmid TraI protein (DNA helicase I) plays an essential role in conjugative DNA transfer as both a transesterase and a helicase. Previous work has shown that the 192-kDa TraI protein is a highly processive helicase, catalytically separating >850 bp under steady-state conditions. In this report, we examine the kinetic mechanism describing DNA unwinding of TraI. The kinetic step size of TraI was measured under both single turnover and pre-steady-state conditions. The resulting kinetic step-size estimate was ∼6-8 bp step-1. TraI can separate double-stranded DNA at a rate of ∼1100 bp s-1, similar to the measured unwinding rate of the RecBCD helicase, and appears to dissociate very slowly from the 3′ terminus following translocation and strand-separation events. Analyses of pre-steady-state burst amplitudes indicate that TraI can function as a monomer, similar to the bacteriophage T4 helicase, Dda. However, unlike Dda, TraI is a highly processive monomeric helicase, making it unique among the DNA helicases characterized thus far.

MutL-catalyzed ATP Hydrolysis Is Required at a Post-UvrD Loading Step in Methyl-directed Mismatch Repair *

Methyl-directed mismatch repair is a coordinated process that ensures replication fidelity and genome integrity by resolving base pair mismatches and insertion/deletion loops. This post-replicative event involves the activities of several proteins, many of which appear to be regulated by MutL. MutL interacts with and modulates the activities of MutS, MutH, UvrD, and perhaps other proteins. The purified protein catalyzes a slow ATP hydrolysis reaction that is essential for its role in mismatch repair. However, the role of the ATP hydrolysis reaction is not understood. We have begun to address this issue using two point mutants: MutL-E29A, which binds nucleotide but does not catalyze ATP hydrolysis, and MutL-D58A, which does not bind nucleotide. As expected, both mutants failed to complement the loss of MutL in genetic assays. Purified MutL-E29A protein interacted with MutS and stimulated the MutH-catalyzed nicking reaction in a mismatch-dependent manner. Importantly, MutL-E29A stimulated the loading of UvrD on model substrates. In fact, stimulation of UvrD-catalyzed unwinding was more robust with MutL-E29A than the wild-type protein. MutL-D58A, on the other hand, did not interact with MutS, stimulate MutH-catalyzed nicking, or stimulate the loading of UvrD. We conclude that ATP-bound MutL is required for the incision steps associated with mismatch repair and that ATP hydrolysis by MutL is required for a step in the mismatch repair pathway subsequent to the loading of UvrD and may serve to regulate helicase loading.