Biology subjects

Lohman, T. M.

Publications and source records attributed to Lohman, T. M..

14 recordsLinked to original sources

bioRxiv2026

Two heads are better than one: Single stranded DNA translocation of UvrD-family dimers vs. monomers

UvrD-family Superfamily 1A helicases are processive ATP-dependent motor proteins that function during DNA replication, recombination, repair, and transcription. UvrD-family monomers translocate along single stranded (ss) DNA with 3-to-5 directionality but must be activated by dimerization to become helicases in the absence of force or accessory factors. Mycobacterium tuberculosis (Mtb) UvrD1 helicase forms dimers via a disulfide bond between native cysteines in the 2B sub-domains of each monomer. E. coli UvrD forms non-covalent dimers using the same 2B domain interface as in Mtb UvrD1. Using both ensemble and single DNA molecule approaches we examined an E. coli UvrD variant (R421C), which forms covalent dimers with constitutive helicase activity. For the first time this has enabled us to compare the ssDNA translocation and helicase activities of covalent dimers and monomers. Crosslinked UvrD dimers exhibit much higher ssDNA translocation processivities than monomers, although with similar translocation rates. Crosslinked UvrD dimers also show highly processive DNA unwinding of thousands of base pairs, much higher than non-crosslinked UvrD dimers, while monomers show no DNA unwinding activity. DNA unwinding rates of crosslinked UvrD dimers are only [~]20% slower than ssDNA translocation rates, indicating they are "active" helicases that directly facilitate duplex destabilization.

biophysics↗

bioRxiv2026

A Conserved Mechanism for Dimerization and Activation of Superfamily 1A UvrD-family Helicases

DNA helicases are ATP-dependent motor proteins that catalyze duplex DNA unwinding and are involved in DNA repair, recombination and replication restart. Prominent members of the non-hexameric SF1A UvrD-family helicases are E. coli UvrD, Rep, B. stearothermophilus PcrA and M. tuberculosis UvrD1. SF1A monomers are processive 3 to 5 single stranded DNA translocases, but need to be activated to become DNA helicases. One mechanism of activation is dimerization. Whereas Rep, UvrD and PcrA form non-covalent dimers, the Mtb UvrD1 helicase forms a redox-dependent covalent dimer. Dimerization of Mtb UvrD1 occurs between the same regulatory domain (2B) within each subunit stabilized by a disulfide bond formed between the same cysteine (Cys451) within each subunit. Dimerization relieves an inhibitory interaction between the 2B domain and duplex DNA within the monomer-DNA complex. We show here that Rep, UvrD and PcrA dimerize using the same 2B-2B interface. By placing a Cys residue within the 2B domains of Rep, UvrD and PcrA in the structurally equivalent position occupied by Cys451 of Mtb UvrD1, all three enzymes form redox-dependent covalent dimers that are constitutively active helicases with increased processivity compared to the non-covalent dimers. Hence, the 2B domain is a general dimerization domain for UvrD-family SF1A helicases.

biochemistry↗

bioRxiv2025

The Nuclease Domain of E. coli RecBCD Regulates DNA Binding and Base Pair Melting

E. coli RecBCD, a hetero-trimeric helicase and nuclease, functions in double stranded (ds) DNA break repair and in degrading foreign DNA. RecBCD possesses ATPase motor domains within both RecB (3 to 5) and RecD (5 to 3) and a nuclease domain within RecB (RecBNuc). RecBCD binds to double stranded DNA ends and initiates DNA unwinding by first melting several DNA base pairs (bp) using only its binding free energy. The RecBNuc domain is docked [~]70 [A] from the duplex DNA binding site in RecBCD-DNA structures but appears to be dynamic and able to move from its docked position. Here, we compare DNA binding of RecBCD and a variant, RecB{Delta}NucCD, in which the 30 kDa nuclease domain has been deleted. RecBCD binding to a blunt DNA end is enthalpically unfavorable and entropically driven. Deletion of RecBNuc results in an increase in DNA binding affinity, suggesting an allosteric effect of RecBNuc. RecB{Delta}NucCD binding to DNA possessing fully pre-melted DNA ends is associated with a large favorable {Delta}Hobs, but much smaller than observed for RecBCD, suggesting that deletion of RecBNuc limits bp melting from a blunt DNA. We also solved cryo-EM structures showing only 4 bp melted upon RecB{Delta}NucCD binding to a blunt ended DNA duplex, less than the 11 bp melted upon RecBCD binding. Thus, the RecB nuclease domain regulates the extent of bp melting by RecBCD. These results also suggest that RecBNuc may manifest its long-range allosteric effect on DNA binding and DNA melting via linker-linker interactions between RecB and RecC.

biophysics↗

bioRxiv2024

Structural Basis for Dimerization and Activation of UvrD-family Helicases

UvrD-family helicases are superfamily 1A motor proteins that function during DNA replication, recombination, repair, and transcription. UvrD family monomers translocate along single stranded (ss) DNA but need to be activated by dimerization to unwind DNA in the absence of force or accessory factors. However, prior structural studies have only revealed monomeric complexes. Here, we report the first structures of a dimeric UvrD-family helicase, Mycobacterium tuberculosis UvrD1, both free and bound to a DNA junction. In each structure, the dimer interface occurs between the 2B subdomains of each subunit. The apo UvrD1 dimer is observed in symmetric compact and extended forms indicating substantial flexibility. This symmetry is broken in the DNA-bound dimer complex with leading and trailing subunits adopting distinct conformations. Biochemical experiments reveal that the E. coli UvrD dimer shares the same 2B-2B interface. In contrast to the dimeric structures, an inactive, auto-inhibited UvrD1 DNA-bound monomer structure reveals 2B subdomain-DNA contacts that are likely inhibitory. The major re-orientation of the 2B subdomains that occurs upon UvrD1 dimerization prevents these duplex DNA interactions, thus relieving the auto-inhibition. These structures reveal that the 2B subdomain serves a major regulatory role rather than participating directly in DNA unwinding.

biochemistry↗

bioRxiv2024

Subunit Communication within Dimeric SF1 DNA Helicases

Monomers of the Superfamily (SF) 1 helicases, E. coli Rep and UvrD, can translocate directionally along single stranded (ss) DNA, but must be activated to function as helicases. In the absence of accessory factors, helicase activity requires Rep and UvrD homo-dimerization. The ssDNA binding sites of SF1 helicases contain a conserved aromatic amino acid (Trp250 in Rep and Trp256 in UvrD) that stacks with the DNA bases. Here we show that mutation of this Trp to Ala eliminates helicase activity in both Rep and UvrD. Rep(W250A) and UvrD(W256A) can still dimerize, bind DNA, and monomers still retain ATP-dependent ssDNA translocase activity, although with lower processivity than wild type monomers. Although neither wtRep monomers nor Rep(W250A) monomers possess helicase activity by themselves, using both ensemble and single molecule methods, we show that helicase activity is achieved upon formation of a Rep(W250A)/wtRep hetero-dimer. An ATPase deficient Rep monomer is unable to activate a wtRep monomer indicating that ATPase activity is needed in both subunits of the Rep hetero-dimer. We find the same results with E. coli UvrD and its equivalent mutant (UvrD(W256A)). Importantly, Rep(W250A) is unable to activate a wtUvrD monomer and UvrD(W256A) is unable to activate a wtRep monomer indicating that specific dimer interactions are required for helicase activity. We also demonstrate subunit communication within the dimer by virtue of Trp fluorescence signals that only are present within the Rep dimer, but not the monomers. These results bear on proposed subunit switching mechanisms for dimeric helicase activity.

biochemistry↗

bioRxiv2023

E. coli RecBCD Nuclease Domain Regulates Helicase Activity but not Single Stranded DNA Translocase Activity

Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as a consequence of mechanically pulling the DNA duplex across a wedge domain in the helicase by the single stranded (ss)DNA translocase activity of the ATPase motors, biochemical data indicate that processive DNA unwinding by the E. coli RecBCD helicase can occur in the absence of ssDNA translocation of the canonical RecB and RecD motors. Here, we present evidence that dsDNA unwinding is not a simple consequence of ssDNA translocation by the RecBCD motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecB{Delta}NucCD) unwinds dsDNA at significantly slower rates than RecBCD, while the rate of ssDNA translocation is unaffected. This effect is primarily due to the absence of the nuclease domain and not the absence of the nuclease activity, since a nuclease-dead mutant (RecBD1080ACD), which retains the nuclease domain, showed no significant change in rates of ssDNA translocation or dsDNA unwinding relative to RecBCD on short DNA substrates ([≤] 60 base pairs). This indicates that ssDNA translocation is not rate-limiting for DNA unwinding. RecB{Delta}NucCD also initiates unwinding much slower than RecBCD from a blunt-ended DNA, although it binds with higher affinity than RecBCD. RecB{Delta}NucCD also unwinds DNA [~]two-fold slower than RecBCD on long DNA ([~]20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecBD1080ACD unwinding are intermediate between RecBCD and RecB{Delta}NucCD. Surprisingly, significant pauses occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, rather than DNA translocation, possibly allosterically. Since the rate of DNA unwinding by RecBCD also slows after it recognizes a chi sequence, RecB{Delta}NucCD may mimic a post-chi state of RecBCD.

biophysics↗

bioRxiv2023

Mycobacterium tuberculosis Ku stimulates multi-round DNA unwinding by UvrD1 monomers

Mycobacterium tuberculosis is the causative agent of Tuberculosis. During the host response to infection, the bacterium is exposed to both reactive oxygen species and nitrogen intermediates that can cause DNA damage. It is becoming clear that the DNA damage response in Mtb and related actinobacteria function via distinct pathways as compared to well-studied model bacteria. For example, we have previously shown that the DNA repair helicase UvrD1 is activated for processive unwinding via redox-dependent dimerization. In addition, mycobacteria contain a homo-dimeric Ku protein, homologous to the eukaryotic Ku70/Ku80 dimer, that plays roles in double-stranded break repair via non-homologous end-joining. Ku has been shown to stimulate the helicase activity of UvrD1, but the molecular mechanism, as well as which redox form of UvrD1 is activated, is unknown. We show here that Ku specifically stimulates multi-round unwinding by UvrD1 monomers which are able to slowly unwind DNA, but at rates 100-fold slower than the dimer. We also demonstrate that the UvrD1 C-terminal Tudor domain is required for the formation of a Ku-UvrD1 protein complex and activation. We show that Mtb Ku dimers bind with high nearest neighbor cooperativity to duplex DNA and that UvrD1 activation is observed when the DNA substrate is bound with two or three Ku dimers. Our observations reveal aspects of the interactions between DNA, Mtb Ku, and UvrD1 and highlight the potential role of UvrD1 in multiple DNA repair pathways through different mechanisms of activation. Declaration of Interests None

biophysics↗

bioRxiv2022

Helicase Activity Promoted Through Dynamic Interactions Between a ssDNA Translocase and a Diffusing SSB Protein

Replication protein A (RPA) is a eukaryotic single stranded (ss) DNA binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of transiently disrupting short regions of duplex DNA by diffusing from a ssDNA that flanks the duplex DNA. Using single molecule total internal reflection fluorescence and optical trapping combined with fluorescence approaches we show that S. cerevisiae Pif1 can use its ATP-dependent 5 to 3 translocase activity to chemo-mechanically push a single human RPA (hRPA) directionally along ssDNA at rates comparable to those of Pif1 translocation alone. We further show that using its translocation activity Pif1 can push hRPA from a ssDNA loading site into a duplex DNA causing stable disruption of at least 9 bp of duplex DNA. These results highlight the dynamic nature of hRPA enabling it to be readily reorganized even when bound tightly to ssDNA and demonstrate a new mechanism by which directional DNA unwinding can be achieved through the combined action of a ssDNA translocase that pushes an SSB protein.

biophysics↗

bioRxiv2022

How Glutamate Promotes Liquid-liquid Phase Separation and DNA Binding Cooperativity of E. coli SSB Protein

E. coli single-stranded-DNA binding protein (EcSSB) displays nearest-neighbor (NN) and non-nearest-neighbor (NNN)) cooperativity in binding ssDNA during genome maintenance. NNN cooperativity requires the intrinsically-disordered linkers (IDL) of the C-terminal tails. Potassium glutamate (KGlu), the primary E. coli salt, promotes NNN-cooperativity, while KCl inhibits it. We find that KGlu promotes compaction of a single polymeric SSB-coated ssDNA beyond what occurs in KCl, indicating a link of compaction to NNN-cooperativity. EcSSB also undergoes liquid-liquid phase separation (LLPS), inhibited by ssDNA binding. We find that LLPS, like NNN-cooperativity, is promoted by increasing [KGlu] in the physiological range, while increasing [KCl] and/or deletion of the IDL eliminate LLPS, indicating similar interactions in both processes. From quantitative determinations of interactions of KGlu and KCl with protein model compounds, we deduce that the opposing effects of KGlu and KCl on SSB LLPS and cooperativity arise from their opposite interactions with amide groups. KGlu interacts unfavorably with the backbone (especially Gly) and side chain amide groups of the IDL and therefore promotes amide-amide interactions in LLPS and NNN-cooperativity. By contrast, KCl interacts favorably with these amide groups and therefore inhibits LLPS and NNN-cooperativity. These results highlight the importance of salt interactions in regulating the propensity of proteins to undergo LLPS.

biochemistry↗

bioRxiv2021

Mycobacterium tuberculosis DNA-repair helicase UvrD1 is activated by redox-dependent dimerization via a conserved 2B domain cysteine.

Mycobacterium tuberculosis (Mtb) causes Tuberculosis and, during infection, is exposed to reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) from the host immune response that can cause DNA damage. UvrD-like proteins are involved in DNA repair and replication and belong to the SF1 family of DNA helicases that use ATP hydrolysis to catalyze DNA unwinding. In Mtb, there are two UvrD-like enzymes where UvrD1 is most closely related to other family members. Previous studies have suggested that UvrD1 is exclusively monomeric, however it is well-known that E. coli UvrD and other UvrD-family members exhibit monomer-dimer equilibria and unwind as dimers in the absence of accessory factors. Here, we reconcile these incongruent studies by showing that Mtb UvrD1 exists in monomer, dimer, and tetramer oligomeric forms where dimerization is regulated by redox potential. We identify a 2B domain cysteine, conserved in many Actinobacteria, that underlies this effect. We also show that UvrD1 DNA unwinding activity correlates specifically with the dimer population and is thus titrated directly via increasing positive (i.e. oxidative) redox potential. Consistent with the regulatory role of the 2B domain and the dimerization-based activation of DNA unwinding in UvrD-family helicases, these results suggest that UvrD1 is activated under oxidizing conditions when it may be needed to respond to DNA damage during infection.

biochemistry↗

bioRxiv2021

Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

biophysics↗

bioRxiv2020

Probing E. coli SSB Protein-DNA topology by reversing DNA backbone polarity

E. coli single strand (ss) DNA binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homo-tetramers bind ssDNA in two major modes differing in occluded site size and cooperativity. The (SSB)35 mode in which ssDNA wraps on average around two subunits is favored at low [NaCl] and high SSB to DNA ratios and displays high "unlimited", nearest-neighbor cooperativity forming long protein clusters. The (SSB)65 mode, in which ssDNA wraps completely around four subunits of the tetramer, is favored at higher [NaCl] (> 200 mM) and displays "limited" low cooperativity. Crystal structures of E. coli SSB and P. falciparum SSB show ssDNA bound to the SSB subunits (OB-folds) with opposite polarities of the sugar phosphate backbones. To investigate whether SSB subunits show a polarity preference for binding ssDNA, we examined EcSSB and PfSSB binding to a series of (dT)70 constructs in which the backbone polarity was switched in the middle of the DNA by incorporating a reverse polarity (RP) phosphodiester linkage, either 3-3 or 5-5. We find only minor effects on the DNA binding properties for these RP constructs, although (dT)70 with a 3-3 polarity switch shows decreased affinity for EcSSB in the (SSB)65 mode and lower cooperativity in the (SSB)35 mode. However, (dT)70 in which every phosphodiester linkage is reversed, does not form a completely wrapped (SSB)65 mode, but rather binds EcSSB in the (SSB)35 mode, with little cooperativity. In contrast, PfSSB, which binds ssDNA only in an (SSB)65 mode and with opposite backbone polarity and different topology, shows little effect of backbone polarity on its DNA binding properties. We present structural models suggesting that strict backbone polarity can be maintained for ssDNA binding to the individual OB-folds if there is a change in ssDNA wrapping topology of the RP ssDNA. Statement of SignificanceSingle stranded (ss) DNA binding (SSB) proteins are essential for genome maintenance. Usually homo-tetrameric, bacterial SSBs bind ssDNA in multiple modes, one of which involves wrapping 65 nucleotides of ssDNA around all four subunits. Crystal structures of E. coli and P. falciparum SSB-ssDNA complexes show ssDNA bound with different backbone polarity orientations raising the question of whether these SSBs maintain strict backbone polarity in binding ssDNA. We show that both E. coli and P. falciparum SSBs can still form high affinity fully wrapped complexes with non-natural DNA containing internal reversals of the backbone polarity. These results suggest that both proteins maintain a strict backbone polarity preference, but adopt an alternate ssDNA wrapping topology.

biophysics↗

bioRxiv2020

Comparative analysis of CPI-motif regulation of biochemical functions of actin capping protein

The heterodimeric actin capping protein (CP) is regulated by a set of proteins that contain CP-interacting (CPI) motifs. Outside of the CPI motif, the sequences of these proteins are unrelated and distinct. The CPI motif and surrounding sequences are conserved within a given protein family, when compared to those of other CPI-motif protein families. Using biochemical assays with purified proteins, we compared the ability of CPI-motif-containing peptides from different protein families to a) bind to CP, b) allosterically inhibit barbed-end capping by CP, and c) allosterically inhibit interaction of CP with V-1, another regulator of CP. We found large differences in potency among the different CPI-motif-containing peptides, and the different functional assays showed different orders of potency. These biochemical differences among the CPI-motif peptides presumably reflect interactions between CP and CPI-motif peptides involving amino-acid residues that are conserved but are not part of the strictly defined consensus, as it was originally identified in comparisons of sequences of CPI motifs(1, 2) across all protein families (1, 2). These biochemical differences may be important for conserved distinct functions of CPI-motif protein families in cells with respect to the regulation of CP activity and actin assembly near membranes.

biochemistry↗

bioRxiv2019

A Novel Antenna Protein Complex in the Life Cycle of Cyanobacterial Photosystem II

In oxygenic photosynthetic organisms, photosystem II (PSII) is a unique membrane protein complex that catalyzes light-driven oxidation of water. PSII undergoes frequent damage due to its demanding photochemistry. However, many facets of its repair and reassembly following photodamage remain unknown. We have discovered a novel PSII subcomplex that lacks five key PSII core reaction center polypeptides: D1, D2, PsbE, PsbF, and PsbI. This pigment-protein complex does contain the PSII core antenna proteins CP47 and CP43, as well as most of their associated low-molecular-mass subunits, and the assembly factor Psb27. Immunoblotting analysis, multiple mass spectrometry techniques, and ultrafast spectroscopic results supported the absence of a functional reaction center in this chlorophyll-protein complex. We therefore refer to it as the no reaction center complex (NRC). Additionally, genetic deletion of PsbO on the PSII lumenal side resulted in an increased NRC population, indicative of a faulty PSII repair scheme at the cellular level. Analytical ultracentrifugation studies and clear native acrylamide gel analysis showed that the NRC complex is a stable pigment-protein complex and not a mixture of free CP47 and CP43 proteins. Our finding challenges the current model of the PSII repair cycle and implies an alternative PSII repair strategy. We propose that formation of this pigment-protein complex maximizes PSII repair economy by preserving an intact PSII core antenna shell in a single complex that is available for PSII reassembly, thus minimizing the risk of randomly diluting multiple recycling components in the thylakoid membrane following a photodamage event at the RC.\n\nSignificance statementPhotosystem II (PSII) converts sunlight into chemical energy, powering nearly all life on Earth. The efficiency of this process is maximized under various environmental conditions by a frequent repair and reassembly cycle that follows inevitable PSII damage even during normal oxygenic photosynthesis. We have isolated a novel pigment protein PSII subcomplex in which, surprisingly, the reaction center (RC) components of PSII are absent. Formation of this stable chlorophyll-protein complex suggests a protective mechanism whereby longer-lived PSII subunits are unplugged from the damaged RC to prevent harmful, aberrant photochemistry during RC repair. This finding provides intriguing new insight into how PSII is assembled and rebuilt to optimize its performance to optimally catalyze one of the most challenging reactions in biology.

plant biology↗