helicase image

Timothy M. Lohman

Marvin A. Brennecke Professor of Biological Chemistry

Department of Biochemistry and Molecular Biophysics
Washington University School of Medicine, St. Louis, MO
 
Molecular Biophysics Program

Biochemistry Program

2801 North Building
Box 8231
office: 314-362-4393
lab: 314-362-4394
FAX: 314-362-7183
E-mail: lohman@biochem.wustl.edu


Research in the Lohman laboratory focuses on obtaining a molecular understanding of the factors that affect the stability, specificity of macromolecular assemblies and protein-nucleic acid complexes in particular, as well as their mechanisms of interaction. Biophysical, biochemical, structural and molecular biological approaches are used to probe these interactions at the molecular level. We are currently investigating two classes of DNA binding proteins: helicases/DNA translocases (motor proteins) and single stranded DNA binding (SSB) proteins. A major emphasis is on the use of equilibrium binding (thermodynamic), pre-steady state kinetic (fluorescence stopped-flow and quenched-flow) and single molecule (fluorescence and optical tweezers) approaches to probe the mechanisms of these protein-DNA interactions.


Research Programs

 

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).

Methods

Transient Kinetics (Stopped-flow and Quenched-flow)

Calorimetry (ITC and DSC)

Analytical Ultracentrifugation

Fluorescence

Single Molecule Fluorescence (TIRF)

 

Publications:

Helicases and DNA Translocases

Reviews:


"Non-hexameric DNA Helicases and Translocases: Mechanisms and Regulation", T. M. Lohman, E. J. Tomko and C. G. Wu, Nat Rev Mol Cell Biol (2008) 9, 391-401. [PDF]

“DNA Helicases: Dimeric Enzyme Action” T. M. Lohman (2004) Encyclopedia of Biological Chemistry, W. J. Lennarz and M. D. Lane, eds. Elsevier Science. [PDF]

"DNA Helicases, Motors that Move Along Nucleic Acids: Lessons from the SF1 Helicase Superfamily", T. M. Lohman, J. Hsieh, N. K. Maluf, W. Cheng, A. L. Lucius, C. J. Fischer, K. M. Brendza, S. Korolev & G. Waksman (2003). The Enzymes, vol XXIII, "ATP and Molecular Motors", ed. F. Tamanoi and D. D. Hackney (Academic Press), pp. 303-369. [PDF]

"Staying on Track: Common Features of DNA Helicases and Microtubule Motors", T. M. Lohman, K. Thorn and R. D. Vale, Cell (1998) 93 9-12. [PDF]

"Mechanisms of Helicase-Catalyzed DNA Unwinding", T. M. Lohman and K.P. Bjornson, Annual Review of Biochemistry (1996) 65, 169-214. [PDF]

"Helicase-catalyzed DNA Unwinding: Energy Coupling by DNA Motor Proteins", K. J. M. Moore and T. M. Lohman, Biophys. J. (1995) 68, 180s-185s.

"Helicase-catalyzed DNA Unwinding", T. M. Lohman, J. Biological Chemistry (1993) 268, 2269-2272. [PDF]

"E.coli DNA Helicases: Mechanisms of DNA Unwinding", T. M. Lohman, Molecular Microbiology (1992) 6, 5-14.


Selected Publications:


"Srs2 Disassembles Rad51 Filaments by a Protein-Protein Interaction Triggering ATP Turnover and Dissociation of Rad51 from DNA", E. Antony, E.J. Tomko, Q. Xiao, L. Krejci, T.M. Lohman and T. Ellenberger, Mol Cell (2009) 35, 105-115. [PDF]

"Influence of DNA End Structure on the Mechanism of Initiation of DNA Unwinding by the Escherichia coli RecBCD and RecBC Helicases", C. J. Wu and T. M. Lohman, J Mol Biol (2008) 382, 312-326. [PDF]

"Kinetic Control of Mg(2+)-dependent Melting of Duplex DNA Ends by Escherichia coli RecBC", C. J. Wong and T. M. Lohman, J Mol Biol (2008) 378, 759-775. [PDF]

“B. stearothermophilus PcrA Monomer is a Single Stranded DNA Translocase but not a Processive Helicase in vitro”. A. Niedziela-Majka, M. A. Chesnik, E. J. Tomko & T. M. Lohman (2007) J. Biological Chemistry 282, 27076-27085. [PDF]

 

"A nonuniform stepping mechanism for E. coli UvrD monomer translocation along single-stranded DNA" Tomko, E.J., Fischer, C.J., Niedziela-Majka, A. and Lohman, T.M., Mol Cell (2007) 26 335-347. [PDF] [Supplement]

"Probing 3'-ssDNA loop formation in E. coli RecBCD/RecBC-DNA complexes using non-natural DNA: A model for "chi" recognition complexes.", Wong, C.J., Rice, R.L., Baker, N.A., Ju, T. and Lohman, T.M., J Mol Biol (2006) 362, 26-43. [PDF]

"Repetitive shuttling of a motor protein on DNA," S. Myong, I. Rasnik, C. Joo, T. M. Lohman and T. Ha. Nature (2005) 437, 1321-1325. [PDF]

"Energetics of DNA End Binding by E.coli RecBC and RecBCD Helicases Indicate Loop Formation in the 3'-Single-stranded DNA Tail " J. C. Wong, A. L. Lucius and T. M. Lohman. J Mol Biol (2005) 352, 765-782. [PDF]

"Autoinhibition of Escherichia coli Rep monomer helicase activity by its 2B subdomain" K.M. Brendza, W. Cheng, C.J. Fischer, M.A. Chesnick, A. Niedziela-Majka and T.M. Lohman. PNAS (2005) 102, 10076-10081. [PDF]

"ATP-dependent translocation of proteins along single-stranded DNA: Models and methods of analysis of pre-steday state kinetics." C. J. Fischer and T. M. Lohman. J Mol Biol (2004) 344, 1265-1286. [PDF]

"Mechanism of ATP-dependent translocation of E. coli UvrD monomers along single-stranded DNA." C. J. Fischer, N. K. Maluf and T. M. Lohman. J Mol Biol (2004) 344, 1287-1309. [PDF]

"Effects of temperature and ATP on the kinetic mechanism and kinetic step-size for E. Coli RecBCD helicase-catalyzed DNA unwinding.", A. L. Lucius and T. M. Lohman. J Mol Biol (2004) 339, 751-771. [PDF]

"Fluorescence stopped-flow studies of single turnover kinetics of E. coli RecBCD helicase-catalyzed DNA unwinding", A.L. Lucius, J. C. Wong and T. M. Lohman. J Mol Biol (2004) 339, 731-750. [PDF]

"Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy", M. C. Murphy, I. Rasnik, W. Cheng, T. M. Lohman, and T. Ha. Biophys. J. (2004) 86, 2530-2537. [PDF]

"DNA-binding orientation and domain conformation of the E. coli rep helicase monomer bound to a partial duplex junction: Single-molecule studies of fluorescently labeled enzymes", I. Rasnik, S. Myong, T. M. Lohman, T. Ha J Mol Biol (2004) 336, 395-408. [PDF]

"General methods for analysis of sequential "n-step kinetic mechanisms: Application to single turnover kinetics of helicase-catalyzed DNA unwinding", A. L. Lucius, N. K. Maluf, C. J. Fischer, T. M. Lohman Biophys. J. (2003) 84, 2224-2239. [PDF]

"Kinetic mechanism for formation of the active, dimeric UvrD helicase-DNA complex", N. K. Maluf, J. A. Ali, T. M. Lohman, J. Biol. Chem. (2003) 278, 31930-31940. [PDF]

"A Dimer of E. coli UvrD is the Active From of the helicase in vitro", N. K. Maluf, C. J. Fischer, T. M. Lohman, J. Mol. Biol. (2003) 325, 913-935. [PDF]

"Self Association Equilbria of E. coli UvrD Helicase Studied by Analytical Ultracentrifugation", N. K. Maluf, T. M. Lohman, J. Mol. Biol. (2003) 325, 889-912. [PDF]

"Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase.", T. Ha, I. Rasnik, W. Cheng, H. P. Babcock, G. H. Gauss, T. M. Lohman and S. Chu, Nature (2002) 419, 638-641. [PDF] [Supplemental]

"DNA unwinding step-size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies.", A. L. Lucius, A. Vindigni, R. Gregorian, J. A. Ali, A. F. Taylor, G. R. Smith and T. M. Lohman J. Mol. Biol. (2002) 324, 409-428. [PDF]

"The 2B domain of E. coli Rep helicase is not required for duplex DNA unwinding activity", W. Cheng, K. M. Brendza, G. H. Gauss, S. Korolev, G. Waksman and T. M. Lohman, Proceedings of the National Academy of Sciences (USA). (2002) 99, 16006-16011. [PDF] [Supplemental]

"E. coli Rep Oligomers are Required to Initiate DNA Unwinding in vitro", W. Cheng, J. Hsieh, K. M. Brendza, T. M. Lohman, J. Mol. Biol. (2001) 310, 327-350. [PDF]

"An Oligomeric Form of E. coli UvrD is Required for Optimal Helicase Activity", J. A. Ali, N. K. Maluf and T. M. Lohman, J. Mol. Biol. (1999) 293, 815-834. [PDF]

"A Two-site Kinetic Mechanism for ATP Binding and Hydrolysis by E. coli Rep Helicase Dimer Bound to a Single Stranded Oligodeoxynucleotide", J. Hsieh, K. J. M. Moore and T. M. Lohman, J. Mol. Biol. (1999) 288, 255-274. [PDF]

"Kinetic Mechanism for the Sequential binding of Two Single-stranded Oligodeoxynucleotides to the E. coli Rep Helicase Dimer", K. P. Bjornson, J. Hsieh, M. Amaratunga and T. M. Lohman, Biochemistry (1998) 37, 891-899. [PDF]

"Comparisons Between the Structures of HCV and Rep Helicases Reveal Structural Similarities Between SF1 and SF2 Superfamilies of Helicases", S. Korolev, N. Yao, T. M. Lohman, P. C. Weber and G. Waksman, Protein Science (1998) 7, 605-610. [PDF]

"Major Domain Swivelling Revealed by the Crystal Structures of Binary and Ternary Complexes of E. coli Rep Helicase Bound to Single Stranded DNA and ADP", S. Korolev, J. Hsieh, G. H. Gauss, T. M. Lohman and G. Waksman, Cell (1997) 90, 635-647. [PDF]

"A Two-site Mechanism for ATP Hydrolysis by the Asymmetric Rep Dimer (P2S) as Revealed by Site-specific Inhibition with ADP-AIF4", I. Wong and T. M. Lohman, Biochemistry (1997) 36, 3115-3125. [PDF]

"Kinetic Measurement of the Step-size of DNA Unwinding by E. coli UvrD Helicase", J. A. Ali and T. M. Lohman, Science (1997) 275, 377-380. [PDF]

"ATP Hydrolysis Stimulates Binding and Release of Single Stranded DNA from Alternating Subunits of the Dimeric E. coli Rep Helicase: Implications for ATP-driven Helicase Translocation", K. P. Bjornson, I. Wong and T. M. Lohman, J. Molecular Biology (1996) 263, 411-422. [PDF]

"ATPase Activity of Escherichia coli Rep Helicase Crosslinked to Single Stranded DNA: Implications for ATP-driven Helicase Translocation", I. Wong and T. M. Lohman, P.N.A.S., U.S.A. (1996) 93, 10051-10056. [PDF]

"ATPase Activity of Escherichia coli Rep Helicase is Dramatically Dependent on its DNA-Ligation and Protein Oligomeric States", I. Wong, K. J. M. Moore, K. P. Bjornson, J. Hsieh and T. M. Lohman, Biochemistry (1996) 35, 5726-5734. [PDF]

"Kinetic Mechanism of DNA Binding and DNA-Induced Dimerization of the Escherichia coli Rep Helicase", K. P. Bjornson, K. J. M. Moore, and T. M. Lohman, Biochemistry (1996) 35, 2268-2282. [PDF]

"Linkage of Protein Assembly to Protein-DNA Binding", I. Wong and T. M. Lohman, Methods in Enzymology, G. K. Ackers and M. Johnson, eds., (1995) 259, 95-127.

"Kinetic Mechanism of Adenine Nucleotide Binding to and Hydrolysis by the E. coli Rep Monomer. I. Use of Fluorescent Nucleotide Analogues", K. J. M. Moore and T. M. Lohman, Biochemistry (1994) 33,14550-14564. [PDF]

"Kinetic Mechanism of Adenine Nucleotide Binding to the E. coli Rep Monomer. II. Application of a Kinetic Competition Approach", K. J. M. Moore and T. M. Lohman, Biochemistry (1994) 33, 14565-14578. [PDF]

"Single-turnover Kinetics of Helicase-catalyzed DNA Unwinding Monitored Continuously by Fluorescence Energy Transfer", K. P. Bjornson, M. Amaratunga, K. J. M. Moore and T. M. Lohman, Biochemistry (1994) 33, 14306-14316. [PDF]

"Hetero-dimer Formation Between Escherichia coli Rep and UvrD Proteins", I. Wong, M. Amaratunga and T. M. Lohman J. Biol. Chem. (1993) 268, 20386-20391. [PDF]

"E.coli Rep Helicase Unwinds DNA by an Active Mechanism", M. Amaratunga and T. M. Lohman, Biochemistry (1993) 32, 6815-6820. [PDF]

"A Double-filter Method for Nitrocellulose Filter Binding:Application to Protein-Nucleic Acid Interactions", I. Wong and T. M. Lohman, Proc. Natl. Acad. Sci., U.S.A. (1993) 90, 5428-5432.

"Kinetics of E. coli Helicase II-catalyzed Unwinding of Fully Duplex and Nicked-circular DNA", G. T. Runyon and T. M. Lohman, Biochemistry (1993) 32,4128-4138. [PDF]

"Overexpression, Purification, DNA Binding and Dimerization of the E. coli uvrD Gene Product (Helicase II)", G. T. Runyon, I. Wong and T. M. Lohman, Biochemistry (1993) 32, 602-612. [PDF]

"Allosteric Effects of Nucleotide Cofactors on E. coli Rep Helicase-DNA Binding", I. Wong and T. M. Lohman, Science (1992) 256, 350-355.

"DNA-induced Dimerization of the E. coli Rep Helicase: Allosteric Effects of Single Stranded and Duplex DNA", I. Wong, K. L. Chao, W. Bujalowski and T. M. Lohman, J. Biological Chemistry (1992) 267, 7596-7610. [PDF]

"DNA-induced Dimerization of the Escherichia coli Rep Helicase", K. Chao and T. M. Lohman, J. Molecular Biology (1991) 221, 1165-1181.

"E.coli Helicase II (UvrD) Protein Initiates DNA Unwinding at Nicks and Blunt-ends", G.T. Runyon, D. G. Bear and T. M. Lohman, Proc. Natl. Acad. Sci., U.S.A. (1990) 87, 6383-6387.

"DNA and Nucleotide-Induced Conformational Changes in the E. coli Rep and Helicase II (UvrD) Proteins", K. Chao and T. M. Lohman, J. Biological Chemistry (1990) 265, 1067-1076. [PDF]

"E. coli Helicase II (UvrD) Protein Can Completely Unwind Fully Duplex Linear and Nicked-Circular DNA", G. T. Runyon and T. M. Lohman, J. Biological Chemistry (1989) 264, 17502-17512. [PDF]

"Large-scale Purification and Characterizationof the E. coli rep Gene Product", T. M. Lohman, K. Chao, S. Sage, J. M. Green and G. T. Runyon, J. Biological Chemistry (1989) 264, 10139-10147. [PDF]


SSB Proteins


Reviews:


"SSB as an Organizer/Mobilizer of Genome Maintenance Complexes", R. D. Shereda, A. G. Kozlov, T. M. Lohman, M. M. Cox and J. L. Keck, CRC Critical Reviews in Biochemistry and Molecular Biology (2008) 43, 289-318. [PDF]


"E. coli Single Stranded DNA Binding Protein: Multiple Binding Modes and Cooperativities", T. M. Lohman and M. E. Ferrari, Annual Review of Biochemistry (1994) 63, 527-570. [PDF]

"E. coli Single Strand Binding Protein: A New Look at Helix Destabilizing Proteins", T. M. Lohman, W. Bujalowski and L. B. Overman, Trends in Biochemical Sciences (1988) 13, 250-255.

"E. coli SSB Protein: Multiple Binding Modes and Cooperativities", T. M. Lohman and W. Bujalowski, The Biology of Nonspecific DNA-Protein Interactions, ed. A. Revzin. CRC Press (1990), 131-168.


Selected Publications:


"SSB protein diffusion on single-stranded DNA stimulates RecA filament formation." Roy, R., Kozlov, A.G., Lohman, T.M. and Ha, T., Nature (2009) 461, 1092-1097. [PDF] | "Slip sliding on DNA" George, N.P. and Keck, J.L. Nature (2009) 461, 1067-1068. [PDF]

"Dynamic structural rearrangements between DNA binding modes of E. coli SSB protein." Roy, R., Kozlov, A.G., Lohman, T.M. and Ha, T., J Mol Biol (2007) 369, 1244-1257. [PDF]

 

"Polar destabilization of DNA duplexes with single-stranded overhangs by the Deinococcus radiodurans SSB protein." Eggington, J.M., Kozlov, A.G., Cox, M.M. and Lohman, T.M., Biochemistry (2006) 45 14490-14502. [PDF]

 

"Saccharomyces cerevisiae replication protein A binds to single-stranded DNA in multiple salt-dependent modes." Kumaran, S., Kozlov, A.G. and Lohman, T.M., Biochemistry (2006) 45 11958-11973. [PDF]

 

"Microsecond dynamics of protein-DNA interactions: Direct observation of the wrapping/unwrapping kinetics of single-stranded DNA around the E. coli SSB tetramer.", Kuznetsov, S.V., Kozlov, A.G., Lohman, T.M. and Ansari, A., J Mol Biol (2006) 359, 55-65. [PDF]

 

"Effects of monovalent anions on a temperature-dependent heat capacity change for Escherichia coli SSB tetramer.", Kozlov, A.G. and Lohman, T.M., Biochemistry (2006) 45, 5190-5205. [PDF]

"The C-terminal domain of full-length E. coli SSB is disordered even when bound to DNA.", Savvides, S.N., Raghunathan, S., Futterer, K., Kozlov, A.G., Lohman, T.M. and Waksman, G., Protein Science (2004) 13, 1942-1947. [PDF]

"Kinetic mechanism of direct transfer of E. coli SSB tetramers between single-stranded DNA molecules.", A. G. Kozlov and T. M. Lohman, Biochemistry (2002) 41, 11611-11627. [PDF]

"Stopped-flow studies of the kinetics of single stranded DNA binding and wrapping around the E. coli SSB tetramer", A. G. Kozlov and T. M. Lohman, Biochemistry (2002) 41, 6032-44. [PDF]

"Structure of the DNA binding domain of E.coli SSB bound to ssDNA", S. Raghunathan, A. Kozlov, T. M. Lohman and G. Waksman, Nature Structural Biology (2000) 7, 648-652. [PDF]

"Large Contributions of Coupled Protonation Equilibria to the Observed Enthalpy and Heat Changes for ssDNA Binding to E. coli SSB Protein", A. G. Kozlov and T. M. Lohman, Proteins: Structure, Function and Genetics (2000) Supplement 4, 8-22. [PDF]

"Adenine Base Unstacking Dominates the Observed Enthalpy and Heat Capacity Changes for E. coli SSB tetramer Binding to Single Stranded Oligoadenylates", A. G. Kozlov and T. M. Lohman, Biochemistry (1999) 38, 10691-10698. [PDF]

"The Importance of Coulombic End Effects: Experimental Characterization of the Effects of Oligonucleotide Flanking Charges on the Strength and Salt-Dependence of Oligocation (L8+) Binding to Single-Stranded DNA Oligomers", W. Zhang, H. Ni, M. W. Capp, C. F. Anderson, T. M. Lohman and M. T. Record, Jr., Biophysical J. (1999) 76, 1008-1017. [PDF]

"Calorimetric Studies of E. coli SSB Protein Single-stranded DNA interactions. Effects of Monovalent Salts on binding Enthalpy", A. G. Korolev and T. M. Lohman, J. Mol. Biol. (1998) 278, 999-1014. [PDF]

"Crystal Structure of the Homo-tetrameric Single Stranded DNA Binding Domain of E. coli Single Stranded DNA Binding Protein Determined by Multiwavelength X-ray Diffraction on the Selenomethionyl Protein at 2.9 Angstrom Resolution", S. Raghunathan, C. S. Ricard, T. M. Lohman and G. Waksman, P.N.A.S., U.S.A. (1997) 94, 6652-6657. [PDF]

"A Mutation in E. coli SSB Protein (W54S) Alters Intra-tetramer Negative Cooperativity and Inter-tetramer Positive Cooperativity for Single Stranded DNA Binding", M. E. Ferrari, J. Fang, and T. M. Lohman, Biophysical Chemistry (1997), 64, 235-251.

"A Highly Salt-dependent Enthalpy Change for E. coli SSB Protein-Nucleic Acid Binding Due to Ion-Protein Interactions", T. M. Lohman, L. B. Overman, M. E. Ferrari and A. G. Kozlov, Biochemistry (1996) 35, 5272-5279. [PDF]

"Apparent Heat Capacity Change Accompanying a Non-specific Protein-DNA Interaction. E. coli SSB Tetramer Binding to Oligodeoxyadenylates", M. E. Ferrari and T. M. Lohman, Biochemistry (1994) 33, 12896-12910. [PDF]

"Cooperative Binding of Escherichia coli SSB Tetramers to Single Stranded DNA in the (SSB)35 Binding Mode", M. E. Ferrari, W. Bujalowski and T. M. Lohman, J. Mol. Biol. (1994) 236, 106-123. [PDF]

"Effects of Base Composition on the Negative Cooperativity and Binding Mode Transitions of E. coli SSB-Single Stranded DNA Complexes", T. M. Lohman and W. Bujalowski, Biochemistry (1994) 33, 6167-6176. [PDF]

"Linkage of pH, Anion and Cation Effects in Protein-Nucleic Acid Equilibria. E. coli SSB Protein-Single Stranded Nucleic Acid Interactions", L. B. Overman and T. M. Lohman, J. Mol. Biol. (1994) 236, 165-178. [PDF]

"Cooperative Binding of Polyamines Induces the Transitions Between E. coli SSB Protein-DNA Binding Modes", T-F. Wei, W. Bujalowski and T. M. Lohman, Biochemistry (1992) 31, 6166-6174.

"Monomers of the Escherichia coli SSB-1 Mutant Protein Bind Single Stranded DNA", W. Bujalowski and T.M. Lohman, J. Molecular Biology (1991) 217, 63-74.

"Monomer-tetramer Equilibrium of the SSB-1 Mutant Single Strand Binding Protein", W. Bujalowski and T. M. Lohman, J. Biological Chemistry (1991) 266, 1616-1626. [PDF]

"Negative Cooperativity in E. coli Single Strand Binding Protein - Oligonucleotide Interactions. I. Evidence and a Quantitative Model", W. Bujalowski and T. M. Lohman, J. Molecular Biology (1989) 207, 249-268.

"Negative Cooperativity in E. coli Single Strand Binding Protein-Oligonucleotide Interactions. II. Salt, temperature and oligonucleotide length effects", W. Bujalowski and T. M. Lohman, J. Molecular Biology (1989) 207, 269-288.

"On the Cooperative Binding of Large Ligands to a One-dimensional Homogeneous Lattice: The Generalized Three-State Lattice Model", W. Bujalowski, T. M. Lohman and C. F. Anderson, Biopolymers (1989) 28, 1637-1643.

"Negative Cooperativity within Individual Tetramers of E. coli SSB Protein is Responsible for the Transition Between the (SSB)35 and (SSB)56 DNA Binding Modes", T. M. Lohman and W. Bujalowski, Biochemistry (1988) 27, 2260-2265. [PDF]

"Equilibrium Binding of E. coli Single Strand Binding Protein to Single Stranded Nucleic Acids in the (SSB)65 Binding Mode. Cation and Anion Effects and Polynucleotide Specificity", L. B. Overman, W. Bujalowski and T. M. Lohman, Biochemistry (1988) 27, 456-471.

"Binding Mode Transitions of E. coli Single Strand Binding Protein-Single Stranded DNA Complexes. Cation, Anion, pH and Binding Density Effects ", W. Bujalowski, L. B. Overman and T. M. Lohman, J. Biological Chemistry (1988) 263, 4629-4640. [PDF]

"Limited Cooperativity in Protein-Nucleic Acid Interactions. A Thermodynamic Model for the Interactions of E. coli Single Strand Binding Protein with Single Stranded Nucleic Acids in the "Beaded", (SSB)65 Mode", W. Bujalowski and T. M. Lohman, J. Molecular Biology (1987) 195, 897-907.

"A General Method of Analysis of Ligand-Macromolecule Equilibria Using a Spectroscopic Signal from the Ligand to Monitor Binding. Application to E. coli SSB Protein-Nucleic Acid Interactions", W. Bujalowski and T. M. Lohman, Biochemistry (1987) 26, 3099-3106.

"E. coli Single Strand Binding Protein Forms Multiple, Distinct Complexes with Single Stranded DNA", W. Bujalowski and T. M. Lohman, Biochemistry (1986) 25, 7799-7802. [PDF]

"Salt Dependent Changes in the DNA Binding Cooperativity of E. coli Single Strand Binding Protein", T. M. Lohman, L. B. Overman and S. Datta, J. Molecular Biology (1986) 187, 603-615.

"Large Scale Overproduction and Rapid Purification of the E. coli ssb Gene Product. Expression of the ssb Gene Under l PL Control", T. M. Lohman, J. M. Green and R. S. Beyer, Biochemistry (1986) 25, 21-25. [PDF]

"Two Binding Modes in E. coli Single Strand Binding Protein (SSB)- Single Stranded DNA Complexes: Modulation by NaCl Concentration", T. M. Lohman and L. B. Overman, J. Biological Chemistry (1985) 260, 3594-3603. [PDF]

"A Model for the Irreversible Dissociation Kinetics of Cooperatively Bound Protein-Nucleic Acid Complexes", T. M. Lohman, Biopolymers (1983) 22, 1697-1713.

"Kinetics and Mechanism of the Association on of the Bacteriophage T4 Gene 32 (Helix Destabilizing) Protein with Single Stranded Nucleic Acids. Evidence for Protein Translocation", T. M. Lohman and S. C. Kowalczykowski, Journal of Molecular Biology (1981) 152, 67-109.

"The Kinetics and Mechanism of Dissociation of Cooperatively Bound T4 Gene32 Protein-Single Stranded Nucleic Acid Complexes. I. Irreversible Dissociation Induced by NaCl Concentration Jumps", T. M. Lohman, Biochemistry (1984) 23, 4656-4665.

"The Kinetics and Mechanism of Dissociation of Cooperatively BoundT4 Gene 32 Protein-Single Stranded Nucleic Acid Complexes. II. Changes in Mechanism as a Function of [NaCl] and other Solution Variables", T. M. Lohman, Biochemistry (1984) 23, 4665-4675.


Thermodynamics and Kinetics of Charged Ligand-Nucleic Acid
Interactions and Nucleic Acid Conformational Transitions


Reviews:


"Thermodynamics of Ligand-Nucleic Acid Interactions", T. M. Lohman and D. P. Mascotti, Methods in Enzymology (1992) 212, 400-424, J. E. Dahlberg and D. M. J. Lilley, eds.

"Non-specific Ligand-DNA Equilibrium Binding Parameters Determined by Fluorescence Methods", T. M. Lohman and D. P. Mascotti, Methods in Enzymology (1992) 212, 424-458, J. E. Dahlberg and J. Lilley, eds.

"Thermodynamic Methods for the Model-Independent Determination of Equilibrium Binding Isotherms for Protein-DNA Interactions, Using Spectroscopic Approaches to Monitor Binding", T. M. Lohman and W. Bujalowski, Methods in Enzymology (1991) 208, 258-290, R. T. Sauer, ed.

"Kinetics of Protein-Nucleic Acid Interactions: Use of Salt Effects to Probe Mechanisms of Interaction", T. M. Lohman, CRC Critical Reviews in Biochemistry (1986) 19, 191-245.

"Thermodynamic Analysis of Ion Effects on the Binding and Conformational Equilibria of Proteins and Nucleic Acids: The Roles of Ion Association or Release,Screening and Ion Effects on Water Activity", M. T. Record, Jr., C. F. Anderson and T. M. Lohman, Quarterly Reviews of Biophysics (1978) 11, 103-178.


Selected Publications:


“Probing Single Stranded DNA Conformational Flexibility Using Fluorescence Spectroscopy”, M. C. Murphy, I. Rasnik, W. Cheng, T. M. Lohman & T. Ha (2004) Biophysical Journal 86, 2530-2537.

"The Importance of Coulombic End Effects: Experimental Characterization of the Effects of Oligonucleotide Flanking Charges on the Strength and Salt-Dependence of Oligocation (L8+) Binding to Single-Stranded DNA Oligomers", W. Zhang, H. Ni, M. W. Capp, C. F. Anderson, T. M. Lohman, M. T. Record, Jr., Biophysical J. (1999) 76, 1008-1017.

"Thermodynamics of Oligoarginines Binding to RNA and DNA", D. P. Mascotti and T. M. Lohman, Biochemistry (1997) 36, 7272-7279. [PDF]

"Large Electrostatic Differences in the Binding Energetics of a Cationic Peptide toOligomeric and Polymeric DNA", W. Zhang, J. P. Bond, C. F. Anderson, T. M. Lohman and M. T. Record, Jr., P.N.A.S., U.S.A.,(1996) 93, 2511-2516. [PDF]

"Thermodynamics of Charged Oligopeptide-Heparin Interactions", D. P. Mascotti and T. M. Lohman, Biochemistry (1995) 34, 2908-2915. [PDF]

"Thermodynamics of Single Stranded RNA and DNA Interactions with Oligolysines Containing Tryptophan. Effects of Base Composition", D. P. Mascotti and T. M. Lohman, Biochemistry (1993) 32, 10568-10579. [PDF]

"Thermodynamics of Single Stranded RNA Binding to Oligolysines Containing Tryptophan", D. P. Mascotti and T. M. Lohman, Biochemistry (1992) 31, 8932-8946. [PDF]

"Thermodynamic Extent of Counterion Release Upon Binding Oligolysines to Single Stranded Nucleic Acids", D. P. Mascotti and T. M. Lohman, Proc. Natl. Acad. Sci., U.S.A. (1990) 87, 3142-3146.

"Use of Difference Boundary Sedimentation Velocity to Investigate Non-specific Protein-Nucleic Acid Interactions", T. M. Lohman, C. G. Wensley, J. Cina, R. R. Burgess and M. T. Record, Jr., Biochemistry (1980) 19, 3516-3522.

"Pentalysine-DNA Interactions: A Model for the General Effects of Ion Concentrations on the Interactions of Proteins with Nucleic Acids", T. M. Lohman, P. L. de Haseth and M. T. Record, Jr., Biochemistry (1980) 19, 3522-3530.

"Nonspecfic Interactions of E. coli RNA Polymerase with Native and Denatured DNA: Differences in the Binding Behavior of Core and Holoenzyme" P. L. de Haseth, T. M. Lohman, R. R. Burgess and M. T. Record, Jr., Biochemistry (1978) 17, 1612-1622.

"An Analysis of Ion Concentration Effects on the Kinetics of Protein-Nucleic Acid Interactions: Application to lac Repressor-Operator Interactions", T. M. Lohman, P. L. de Haseth and M. T. Record, Jr., Biophysical Chemistry (1978) 8, 281-294.

"A Semi-empirical Extension of Polyelectrolyte Theory to the Treatment of Oligo-electrolytes", M. T. Record, Jr. and T. M. Lohman, Biopolymers (1978) 17, 159-166.

"Interpretation of Monovalent and Divalent Ion Effects on the Binding of lac-Repressor to Operator", M. T. Record, Jr., P. L. de Haseth and T. M. Lohman, Biochemistry (1977) 16, 4791-4796.

"The Nonspecific Binding of lac-Repressor to DNA. An Association Reaction Driven by Counterion Release", P. L. de Haseth, T. M. Lohman and M. T. Record, Jr., Biochemistry (1977) 16, 4783-4790.

"Ion Effects on Ligand-Nucleic Acid Interactions", M. T. Record, Jr., T. M. Lohman and P. de Haseth, Journal of Molecular Biology (1976) 107, 145-158.