Research

Research Programs

Helicase-catalyzed DNA Unwinding and ssDNA Translocation
SSB Protein-DNA Interactions

Mechanisms of Helicase-catalyzed DNA Unwinding and ssDNA Translocation

The unwinding of duplex DNA to form single stranded (ss) DNA intermediates is a prerequisite for replication, recombination and repair and this process is catalyzed by a class of enzymes, referred to as helicases. These ubiquitous enzymes utilize nucleoside 5’-triphosphate (e.g., ATP) binding and hydrolysis to destabilize the hydrogen bonds between the complementary base pairs (bp) in duplex DNA. Intimately linked to the DNA unwinding reaction is the requirement for helicases to translocate along the DNA filament in order to unwind DNA processively at rates that can be as fast as 500-1000 bp s^-1. Since DNA helicases transduce the chemical free energy change associated with NTP hydrolysis into mechanical energy to unwind DNA and also translocate along DNA, they are members of the general class of “motor proteins” with which they have several similarities. We are currently investigating three DNA helicases, E.coli Rep, E. coli UvrD (Helicase II) protein and E. coli RecBCD, a heterotrimeric bipolar helicase composed of two translocase subunits (RecB and RecD). These helicases can unwind duplex DNA and translocate along ssDNA in the absence of DNA synthesis and hence provide an excellent system to examine the mechanisms of these processes in vitro. We are interested in understanding the mechanism of helicase-catalyzed DNA unwinding and the molecular details of protein translocation along DNA and the role of ATP in these processes.DNA helicases are ubiquitous enzymes, having been identified in various prokaryotes and eukaryotes as well as in bacteriophages and viruses. Most organisms encode multiple helicases; for example, E. coli encodes at least 12 different helicases. Nucleic acid translocases, not all of which are helicases, use NTP to move with biased directionality along single-stranded (ss) or duplex (ds) nucleic acids. Although DNA helicases were discovered on the basis of their ability to catalyze separation of the complementary strands of dsDNA during replication, recombination and repair of DNA, it is now evident that this class of enzymes also functions in a range of other biological processes, including displacement of proteins from DNA and RNA, ‘remodeling’ of chromatin, movement of Holliday junctions, and the catalysis of a range of nucleic acid conformational changes. The importance of these enzymes is underscored by the numerous human diseases (e.g., xeroderma pigmentosum,Werner’s syndrome, Bloom’s syndrome and Cockayne’s syndrome) that are associated with defective helicases/translocases.Since DNA helicases are essential enzymes in all aspects of DNA metabolism it is important to obtain a detailed molecular understanding of the mechanism(s) by which helicases function. To catalyze the unwinding of duplex DNA, a helicase must cycle, vectorially, through a series of energetic (conformational) states, driven by the binding and/or hydrolysis of NTP and subsequent release of products (NDP + PO₄⁼). Therefore, a molecular understanding of helicase-catalyzed DNA unwinding requires information on the coupling of NTP binding and hydrolysis to DNA unwinding as well as the identification of the intermediate helicase-DNA states that occur during unwinding. This requires quantitative studies of the energetics (thermodynamics) and kinetics of helicase binding to DNA and nucleotide cofactors (NTP, NDP, Pi) as well as structural information.

Studies over the last decade have identified a variety of helicases that differ in both structure and mechanism of unwinding. Helicases were first classified into superfamilies (SF1 and SF2) and families (F3, F4 and F5) on the basis of regions of their primary structure (so-called ‘helicase motifs’). Although still useful, these classifications were made before the availability of structural information and before many of the enzymes had been characterized biochemically. It is now evident that these helicase motifs are present in a wide range of NTP-dependent nucleic acid enzymes, many of which are not helicases, and some of which do not even appear to translocate along nucleic acids. Hence these motifs more generally identify nucleic acid-stimulated NTPases.

The ring-shaped hexameric helicases encircle the nucleic acid and function mainly, but not exclusively, as the primary helicases in chromosomal DNA replication; however, the majority of helicases/translocases are non-hexameric and belong to superfamily (SF)1 or SF2. The two largest superfamilies, SF1 and SF2, are each defined by seven conserved regions of primary structure. My lab focuses on the mechanisms of translocation and duplex DNA unwinding of mainly SF1 enzymes that act processively — i.e., unwind multiple base pairs or translocate multiple bases before dissociating from the DNA.

A complete understanding of the mechanism of nucleic acid unwinding and/or translocation by a motor protein requires information about the oligomeric structure of the functional enzyme, as well as the rate, processivity, directional bias, step size and stoichiometry of ATP coupling. A combination of structural, pre-steady-state kinetic, thermodynamic and single molecule studies is required to address all of these issues.

In ensemble studies, quantitative information about the rate and processivity of unwinding requires pre-steady-state kinetic measurements. Steady-state, multiple turnover experiments do not yield this information as only the net production of strand-separated duplexes is measured, and this rate is limited by the slowest kinetic step(s) in the multiple turnover cycle, which generally involve enzyme binding, dissociation, re-binding, or protein oligomerization. As these processes are usually much slower than the rates of unwinding or translocation, no information about the latter can be obtained from steady-state experiments. By contrast, a pre-steady-state or single cycle unwinding measurement is sensitive to the kinetic steps that occur within the unwinding cycle. Single cycle or single turnover unwinding or translocation assays also provide the most straightforward methods to determine the relative activities of different oligomeric forms of helicases and translocases, although this requires independent information of the oligomeric state of the enzyme that is bound to the DNA substrate at the start of the reaction. In collaboration with Prof. Taekjip Ha (University of Illinois, Urbana), we are using single molecule fluorescence methods to obtain detailed information on the mechanism of translocation and DNA unwinding. These methods have the added benefit that they can detect stochastic processes, such as pausing, reversal of direction and repetitive shuttling that are undetectable in ensemble experiments. We have recently acquired a total internal reflectance fluorescence (TIRF) microscope and are now able to use these single molecule fluorescence approaches in our own lab.

In collaboration with Dr. Gabriel Waksman and Dr. Sergey Korolev, we have solved the x-ray crystal structures of two conformations of a monomer of the E. coli Rep helicase/translocase bound to ssDNA (Korolev et al. (1997) Cell 90, 635). The two forms of this 4 sub-domain protein differ by a 130 degree rotation of one sub-domain (2B) with respect to the other three (see below). Although the full length Rep monomer has rapid and processive 3’ to 5’ ssDNA translocase activity, the 2B sub-domain is auto-inhibitory for Rep monomer helicase activity. In fact, E. coli UvrD monomers and B. stearothermophilus PcrA monomers display this same behavior in vitro, i.e., monomers of these enzymes are rapid and processive and directional (3’ to 5’) ssDNA translocases, yet the monomers display no helicase activity in vitro. Helicase activity of these enzymes needs to be activated either by self-oligomerization or through heterologous interactions with accessory proteins.

SSB Proteins

We are also investigating the E. coli ssb gene product (SSB protein) as well as the eukaryotic replication protein A (RPA), which are essential components in DNA replication, recombination and repair. The E. coli SSB protein is a homotetrameric helix destabilizing protein that binds selectively and cooperatively to ss-DNA and facilitates DNA unwinding by the DNA helicases. These proteins are present in high concentrations in vivo, bind with high specificity to ss-DNA and function, at least in part, by binding to ss-DNA formed transiently during replication, recombination and repair. This class of proteins binds nonspecifically to ss-DNA and in most cases with positive cooperativity. This latter property may be necessary for the biological function of helix destabilizing proteins, since if cooperativity between DNA-bound proteins is sufficiently high, and of the “unlimited” type, these proteins can saturate a stretch of ss-DNA at low protein concentrations. The ability to saturate a long stretch of ss-DNA, which is not possible for proteins that bind noncooperatively, is thought to be necessary to protect the DNA from the action of nucleases, as well as to hold the DNA in a conformation which facilitates the function of other replication, recombination or repair enzymes.

Until recently, SSB proteins have often been described as inert ssDNA coatings that protected the ssDNA. However, recent research has demonstrated a far more complex role of SSB proteins. For example, the E. coli SSB protein is now known to interact with at least 14 other proteins involved in DNA metabolism. Most, if not all, of these interactions occur mainly with the acidic C-terminus of SSB and SSB serves to bind these proteins and bring them to their target locations to function during replication, recombination and repair processes.

The binding of the tetrameric E. coli SSB protein to ssDNA is complex, since it can bind in several distinct binding modes, designated as (SSB)_n, depending on the solution conditions (see cartoon above and Lohman and Ferrari (1994) Annual Review of Biochemistry (1994) 63, 527 for a review). These modes differ in both the number of nucleotides (n) occluded by each bound tetramer as well as in the type and extent of inter-tetramer positive cooperativity. At 25°C (pH 8.1), three binding modes have been identified with n=35±2,56±3, and 65±3 nucleotides per tetramer. The (SSB)₃₅ mode is favored at high SSB binding density and low salt (10 mM NaCl), whereas the higher site size modes are stabilized by higher salt concentrations and at low SSB binding density. Only two of the subunits of the tetramer interact with ss-DNA in the (SSB)₃₅ mode, whereas all four subunits interact with DNA in both the (SSB)₅₆ and (SSB)₆₅ modes. The relative stability of the (SSB)₃₅ mode at low salt is due partly to an extensive negative cooperativity among the DNA binding sites within the SSB tetramer, such that the affinity of ss-DNA binding to the third and fourth subunits of the tetramer decreases dramatically upon lowering the salt concentration. In at least the high site size, beaded mode, the ssDNA wraps completely around the outside of the SSB tetramer. The figure below shows models for the two major SSB binding modes based on x-ray crystal structural studies done in collaboration with Prof. Gabriel Waksman.

A common feature of SSB proteins is their ability to bind with positive cooperativity to ss-polynucleotides and thus form clusters of protein, even at low binding densities. However, the type and magnitude of the positive cooperativity observed for the E. coli SSB tetramer binding to ss-DNA differs dramatically for the (SSB)₆₅ and (SSB)₃₅ modes. SSB tetramers bind with an “unlimited” type of inter-tetramer cooperativity in the (SSB)₃₅ mode, and thus can form long protein clusters which can saturate the DNA, in a manner similar to that observed for the phage T4 gene 32 protein. In contrast, binding in the (SSB)₆₅ mode occurs with a “limited” type of positive inter-tetramer cooperativity, such that protein clustering is limited to the formation of dimers of tetramers (“octamers”). Since the different SSB polynucleotide binding modes display such very different properties, we have proposed that some of the different binding modes may be used selectively in DNA replication, recombination, and repair.

We are currently investigating the molecular interactions that stabilize the different SSB-ssDNA complexes, the factors that influence the distribution of binding modes as well as the different cooperativities in each mode. We are also investigating the specificity of SSB interactions with the myriad of proteins that are known to bind to SSB (14 so far). Our approaches include equilibrium binding (thermodynamic) studies, using fluorescence and isothermal titration calorimetry to monitor the interactions. Stopped-flow fluorescence and single molecule fluorescence techniques are also used to examine the kinetics and mechanism of DNA binding to the tetrameric SSB protein. We are also using single molecule fluorescence approaches in collaboration with Prof. Taekjip Ha (University of Illinois, Urbana) and optical tweezer methods in collaboration with Prof. Yann Chemla (University of Illinois, Urbana).