The Experts below are selected from a list of 1950 Experts worldwide ranked by ideXlab platform
Hungwen Li - One of the best experts on this subject based on the ideXlab platform.
-
Intersubunit Regulation Between Nuclease and Helicase Domains of RecBCD Enzyme
Biophysical Journal, 2020Co-Authors: Jui-yun Chang, Hungwen LiAbstract:The Escherichia coliRecBCD helicase/nuclease initiates homologous recombinational repair of damaged blunt-end duplex DNA molecules. RecBCD, a multifunctional enzyme complex, contains two DNA motors as well as a nuclease domain to process duplex DNA and generate single-stranded DNA molecules. We used single-molecule tethered particle motion (TPM) experiments to investigate the regulation mechanism between the nuclease domain and two helicase domains of RecBCD enzyme using calcium ions, which specifically inhibit nuclease activity. In the absence of calcium ions, RecBCD translocation rate is found to slow down after recognizing chi sequence. However, in the presence of calcium ions, the rate change in individual RecBCD translocation is abolished, returning similar averaged translocation rate before (71 ± 20 bp/s) and post (81 ± 36 bp/s) chi-sequence, under 30μM ATP. Furthermore, large portion of individual RecBCD unwinding time courses (13 out of 32) revealed repetitive forward and backward translocation along individual DNA molecules. Compared with the experiments carried out without calcium ions, the processivity of RecBCD also decreases when the nuclease domain is inhibited. About 50 percent of translocating tethers (17 out of 32) stalled within 1.5 Kb DNA used in the presence of calcium ions. Together, these observations suggest that the nuclease domain, located in the RecB subunit, plays regulatory roles not only in RecBCD translocation properties but also in chi-regulated intersubunit interaction in this complex machine of the RecBCD enzyme.
-
how chi sequence modifies RecBCD single stranded dna translocase activity
ChemPhysChem, 2018Co-Authors: Cinya Chung, Hungwen LiAbstract:: E. coli RecBCD initiates homologous repair as well as degrades foreign DNA. Recognition of chi sequence (5'-GCTGGTGG-3') switches RecBCD from a destructive, nucleolytic mode into a repair-active one that promotes RecA-mediated recombination. RecBCD includes a 3'-to-5' single-stranded DNA (ssDNA) translocase in RecB subunit, a 5'-to-3' translocase in RecD, and a secondary translocase activity associated with RecBC. To understand how chi specifically affects each translocase activity, we directly visualized individual RecBCD translocating along DNA substrates containing a ssDNA gap of different polarities, with or without chi. Disappearance of RecBCD from the ssDNA signals the loss of the ssDNA translocase activity. For substrates containing a ssDNA gap that RecBCD encounters in the 3'-to-5' polarity (3'-to-5' ssDNA), wild-type RecBCD disappears from the DNA substrates with similarly high percentage, either with chi or without. This suggests that (1) the 3'-to-5' translocase in RecB is unaffected by chi and (2) it is low in processivity. With substrates containing a ssDNA gap that RecBCD encounters in the 5'-to-3' polarity (5'-to-3' ssDNA), we found that the leaving percentage increases significantly with chi, implying inactivation of the 5'-to-3' translocase of RecD upon chi recognition. Surprisingly, the RecD defective mutant RecBCDK177Q showed only ≈50 % leaving on 5'-to-3' ssDNA, directly revealing the presence of RecBC secondary translocase and its activity is unaffected by chi. Multiple ssDNA translocases within the RecBCD complex both before and after chi ensures processive unwinding of DNA substrates required for efficient recombination events.
-
sequence dependent nanometer scale conformational dynamics of individual RecBCD dna complexes
Nucleic Acids Research, 2016Co-Authors: Ashley R Carter, Hungwen Li, Thomas T Perkins, Maasa H Seaberg, Christopher J WildsAbstract:: RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2-4 μM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD-DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5' strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket.
-
Sequence-dependent nanometer-scale conformational dynamics of individual RecBCD–DNA complexes
Nucleic Acids Research, 2016Co-Authors: Ashley R Carter, Hungwen Li, Maasa H Seaberg, Christopher J Wilds, Thomas T PerkinsAbstract:: RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2-4 μM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD-DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5' strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket.
-
RecBCD fails to bypass the 5 to 3 single stranded dna gap after translocating along individual chi containing duplex dna
Biophysical Journal, 2014Co-Authors: Cinya Chung, Hungwen LiAbstract:The E. coli RecBCD plays an important role of initiating the repair of double-stranded DNA breaks (DSB). Translocating and recognizing chi sequence (5'-GCTGGTGG-3') have been implicated with a conformational change that enables the enzyme to preserve the 3'-to-5' single-stranded DNA for RecA assembly. RecBCD is composed of three subunits, RecB (3'-to-5' helicase), RecD (5'-to-3' helicase), and RecC. Here we used a single-molecule tethered particle motion technique to directly monitor the translocation of RecBCD along chi-contained DNA molecules. Using bead-labeled enzymes, we monitored the RecBCD translocation along individual DNA by measuring the gradual decrease in the bead Brownian motion as the enzyme moves along the DNA towards the surface. DNA substrates were designed that RecBCD would encounter a single-stranded DNA gap after the recognition of the chi sequence. While translocating along chi-free DNA substrates, the time traces showed no apparent pause, which neither 3'-to-5' nor 5'-to-3' ssDNA gap influents the movement of the enzyme1. However, over 50% of RecBCD enzymes failed to pass through the 5'-to-3' ssDNA gap after translocating over chi-containing duplex DNA. Considering RecD as a major 5'-to-3' ssDNA translocase in the RecBCD complex, our observation is consistent with the model that the conformation change occurs after chi recognition and RecD is disengaged from the 5'-ssDNA.(1) Chung, C.; Li, H. W. J. Am. Chem. Soc. 2013, 135, 8920.
Stephen C Kowalczykowski - One of the best experts on this subject based on the ideXlab platform.
-
Short Article Direct Visualization of RecBCD Movement Reveals Cotranslocation of the RecD Motor after Recognition
2020Co-Authors: Naofumi Handa, Ronald J Baskin, Piero R Bianco, Stephen C KowalczykowskiAbstract:Summary changes elicited persist for the duration of the translo- cation event in cis, but the phenomenon is fully revers- In Escherichia coli , (5 -GCTGGTGG-3 ) is a recombi- ible and catalytic after dissociation of RecBCD from thenation hotspot recognized by the RecBCD enzyme. χ-containing DNA molecule (Dixon et al., 1994). The Recognition of reduces both nuclease activity and molecular basis for this regulation remains unknown. translocation speed of RecBCD and activates RecA- One attractive hypothesis, proposed over a decade loading ability. RecBCD has two motor subunits, ago, is the RecD-ejection model (Koppen et al., 1995; RecB and RecD, which act simultaneously but inde- Kuzminov et al., 1994; Myers et al., 1995; Stahl et al., pendently. A longstanding hypothesis to explain the 1990; Thaler et al., 1989). This longstanding hypothesis changes elicited by interaction has been “ejection” is based on the many similarities in genetic and bio- of the RecD motor from the holoenzyme at . To test
-
RecBCD enzyme and the repair of double stranded dna breaks
Microbiology and Molecular Biology Reviews, 2008Co-Authors: Mark S Dillingham, Stephen C KowalczykowskiAbstract:Summary: The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA, protecting the bacteria from phages and extraneous chromosomal DNA. The RecBCD enzyme is, however, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzyme's vigorous nuclease activity, switches the polarity of the attenuated nuclease activity to the 5′ strand, changes the operation of its motor subunits, and instructs the enzyme to begin loading the RecA protein onto the resultant Chi-containing single-stranded DNA. This enzyme is a prototypical example of a molecular machine: the protein architecture incorporates several autonomous functional domains that interact with each other to produce a complex, sequence-regulated, DNA-processing machine. In this review, we discuss the biochemical mechanism of the RecBCD enzyme with particular emphasis on new developments relating to the enzyme's structure and DNA translocation mechanism.
-
RecBCD enzyme switches lead motor subunits in response to χ recognition
Cell, 2007Co-Authors: Ichiro Amitani, Maria Spies, Ronald J Baskin, Stephen C KowalczykowskiAbstract:Summary RecBCD is a DNA helicase comprising two motor subunits, RecB and RecD. Recognition of the recombination hotspot, χ, causes RecBCD to pause and reduce translocation speed. To understand this control of translocation, we used single-molecule visualization to compare RecBCD to the RecBCD K177Q mutant with a defective RecD motor. RecBCD K177Q paused at χ but did not change its translocation velocity. RecBCD K177Q translocated at the same rate as the wild-type post-χ enzyme, implicating RecB as the lead motor after χ. P1 nuclease treatment eliminated the wild-type enzyme's velocity changes, revealing a χ-containing ssDNA loop preceding χ recognition and showing that RecD is the faster motor before χ. We conclude that before χ, RecD is the lead motor but after χ, the slower RecB motor leads, implying a switch in motors at χ. We suggest that degradation of foreign DNA needs fast translocation, whereas DNA repair uses slower translocation to coordinate RecA loading onto ssDNA.
-
direct visualization of RecBCD movement reveals cotranslocation of the recd motor after χ recognition
Molecular Cell, 2005Co-Authors: Naofumi Handa, Ronald J Baskin, Piero R Bianco, Stephen C KowalczykowskiAbstract:Summary In Escherichia coli , χ (5′-GCTGGTGG-3′) is a recombination hotspot recognized by the RecBCD enzyme. Recognition of χ reduces both nuclease activity and translocation speed of RecBCD and activates RecA-loading ability. RecBCD has two motor subunits, RecB and RecD, which act simultaneously but independently. A longstanding hypothesis to explain the changes elicited by χ interaction has been "ejection" of the RecD motor from the holoenzyme at χ. To test this proposal, we visualized individual RecBCD molecules labeled via RecD with a fluorescent nanoparticle. We could directly see these labeled, single molecules of RecBCD moving at up to 1835 bp/s (∼0.6 μm/s). Those enzymes translocated to χ, paused, and continued at reduced velocity, without loss of RecD. We conclude that χ interaction induces a conformational change, resulting from binding of χ to RecC, and not from RecD ejection. This change is responsible for alteration of RecBCD function that persists for the duration of DNA translocation.
-
homologous recombination by the RecBCD and recf pathways
2005Co-Authors: Maria Spies, Stephen C KowalczykowskiAbstract:Interaction with χ affects the helicase activity of RecBCD enzyme. Recognition of χ causes the enzyme to pause briefly at χ and to resume translocation after the χ site, but at a rate that is reduced by approximately twofold. In response to χ the RecBCD enzyme accomplishes both tasks essential for initiation of homologous recombination: (i) it recesses the double-strand break (DSB) to produce an ssDNA-tailed duplex DNA with χ at its terminus, and (ii) it catalyzes formation of the RecA nucleoprotein filament on the ssDNA produced. Interestingly, the efficiency of conjugational and transductional recombination by the RecF pathway in the recBC sbcBC cells is similar to that of the RecBCD pathway in wild-type cells, showing that the machinery of this pathway can be as productive as that of the RecBCD pathway. The loading of RecA protein is an essential aspect of recombination in the RecBCD pathway. On the other hand, recB recF double mutants are deficient in recombination between chromosomal direct repeats, suggesting that both RecBCD and RecF pathways play major roles in recombination. Homologous recombination can be initiated at either DSBs or single-strand DNA gap (SSG) in duplex DNA. Two major pathways are responsible for homologous recombination in wild-type E. coli: The RecBCD pathway is specific for the recombinational repair of DSBs, and in the wild-type cells, the RecF pathway is primarily used for recombination that initiates at SSGs.
Thomas T Perkins - One of the best experts on this subject based on the ideXlab platform.
-
sequence dependent nanometer scale conformational dynamics of individual RecBCD dna complexes
Nucleic Acids Research, 2016Co-Authors: Ashley R Carter, Hungwen Li, Thomas T Perkins, Maasa H Seaberg, Christopher J WildsAbstract:: RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2-4 μM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD-DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5' strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket.
-
Sequence-dependent nanometer-scale conformational dynamics of individual RecBCD–DNA complexes
Nucleic Acids Research, 2016Co-Authors: Ashley R Carter, Hungwen Li, Maasa H Seaberg, Christopher J Wilds, Thomas T PerkinsAbstract:: RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2-4 μM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD-DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5' strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket.
-
conformational dynamics of single RecBCD molecules
Biophysical Journal, 2010Co-Authors: Hungwen Li, Martha Hosotani, Ashley R Carter, Thomas T PerkinsAbstract:RecBCD is a multifunctional enzyme possessing both helicase and nuclease activities. It harnesses the energy of ATP hydrolysis to processively unwind DNA. We used an optical-trapping assay featuring one base-pair stability to investigate the mechanism of RecBCD unwinding. Records of RecBCD motion at 6 pN of applied load showed fluctuations [4.1 ± 0.1 bp, (mean ± std. err.; freq. bandwidth = 0.1-10 Hz)] substantially above the control records with DNA alone. These fluctuations persisted when the enzyme's forward motion was stopped by removing ATP. Records of RecBCD bound to blunt-end DNA in the absence of ATP showed reduced dynamics (2.4 ± 0.2 bp), indicating the primary origin of the fluctuations was not due to anchoring via RecBCD. Prior biochemical studies showed that unwinding activity is preceded by an initiation phase consisting of several kinetic steps that generates a 10-nt, 5′-tailed substrate inside the RecBCD-DNA complex that engages RecD's helicase domain. This work also showed that binding to a forked 3′-(dT)6 and 5′-(dT)6 DNA substrate is kinetically equivalent to binding to a blunt-end DNA, while a 3′-(dT)6 and 5′-(dT)10substrate bypasses initiation. We found that records of RecBCD bound to these tailed DNA substrates showed fluctuations that quantitatively mirrored our records of RecBCD bound to blunt-end DNA and stopped within a long DNA substrate, respectively. Thus, the onset of large fluctuations in the RecBCD-DNA complex was coincident with that of unwinding activity. The magnitude and frequency of fluctuations increased when the DNA sequence immediately in front of the forked substrate was changed from GC to AT base pairs, consistent with RecBCD transiently translocating along the DNA without ATP hydrolysis. A tightly bound state with reduced dynamics (2.7 ± 0.1 bp) was observed with ADP-BeF2. These findings support a ratchet model for RecBCD movement.
-
Single-molecule studies of RecBCD.
Methods of Molecular Biology, 2009Co-Authors: Thomas T Perkins, Hungwen LiAbstract:: RecBCD is a processive molecular motor composed of two independent helicase domains and a nuclease domain. Understanding the molecular mechanism of its motor activity involves, in part, determining RecBCD's translocation properties (e.g., velocity, propensity to pause, pause duration). Single-molecule techniques, in general, and optical trapping, specifically, provide for measuring the translocation of individual molecules along DNA. We developed a high-spatial resolution optical-trapping assay for RecBCD. The RecBCD is anchored to the surface via a genetically engineered biotin. This RecBCD-bio exhibited native activity, as measured by biochemical assays. Motion is continuous down to a detection limit of 2 nm, implying a unitary step size below 6 base pairs. Unexpectedly, the catalytic rate changes abruptly and persists at different values for tens of seconds. This technically demanding, high-resolution optical-trapping assay is complemented by a simpler single-molecule assay-the tethered particle motion assay.
-
forward and reverse motion of single RecBCD molecules on dna
Biophysical Journal, 2004Co-Authors: Thomas T Perkins, Hungwen Li, Ravindra V Dalal, Jeff Gelles, Steven M BlockAbstract:RecBCD is a processive, DNA-based motor enzyme with both helicase and nuclease activities. We used high-resolution optical trapping to study individual RecBCD molecules moving against applied forces up to 8 pN. Fine-scale motion was smooth down to a detection limit of 2 nm, implying a unitary step size below six basepairs (bp). Episodes of constant-velocity motion over hundreds to thousands of basepairs were punctuated by abrupt switches to a different speed or by spontaneous pauses of mean length 3 s. RecBCD occasionally reversed direction, sliding backward along DNA. Backsliding could be halted by reducing the force, after which forward motion sometimes resumed, often after a delay. Elasticity measurements showed that the DNA substrate was partially denatured during backsliding events, but reannealed concomitant with the resumption of forward movement. Our observations show that RecBCD-DNA complexes can exist in multiple, functionally distinct states that persist for many catalytic turnovers: such states may help tune enzyme activity for various biological functions.
Cinya Chung - One of the best experts on this subject based on the ideXlab platform.
-
how chi sequence modifies RecBCD single stranded dna translocase activity
ChemPhysChem, 2018Co-Authors: Cinya Chung, Hungwen LiAbstract:: E. coli RecBCD initiates homologous repair as well as degrades foreign DNA. Recognition of chi sequence (5'-GCTGGTGG-3') switches RecBCD from a destructive, nucleolytic mode into a repair-active one that promotes RecA-mediated recombination. RecBCD includes a 3'-to-5' single-stranded DNA (ssDNA) translocase in RecB subunit, a 5'-to-3' translocase in RecD, and a secondary translocase activity associated with RecBC. To understand how chi specifically affects each translocase activity, we directly visualized individual RecBCD translocating along DNA substrates containing a ssDNA gap of different polarities, with or without chi. Disappearance of RecBCD from the ssDNA signals the loss of the ssDNA translocase activity. For substrates containing a ssDNA gap that RecBCD encounters in the 3'-to-5' polarity (3'-to-5' ssDNA), wild-type RecBCD disappears from the DNA substrates with similarly high percentage, either with chi or without. This suggests that (1) the 3'-to-5' translocase in RecB is unaffected by chi and (2) it is low in processivity. With substrates containing a ssDNA gap that RecBCD encounters in the 5'-to-3' polarity (5'-to-3' ssDNA), we found that the leaving percentage increases significantly with chi, implying inactivation of the 5'-to-3' translocase of RecD upon chi recognition. Surprisingly, the RecD defective mutant RecBCDK177Q showed only ≈50 % leaving on 5'-to-3' ssDNA, directly revealing the presence of RecBC secondary translocase and its activity is unaffected by chi. Multiple ssDNA translocases within the RecBCD complex both before and after chi ensures processive unwinding of DNA substrates required for efficient recombination events.
-
RecBCD fails to bypass the 5 to 3 single stranded dna gap after translocating along individual chi containing duplex dna
Biophysical Journal, 2014Co-Authors: Cinya Chung, Hungwen LiAbstract:The E. coli RecBCD plays an important role of initiating the repair of double-stranded DNA breaks (DSB). Translocating and recognizing chi sequence (5'-GCTGGTGG-3') have been implicated with a conformational change that enables the enzyme to preserve the 3'-to-5' single-stranded DNA for RecA assembly. RecBCD is composed of three subunits, RecB (3'-to-5' helicase), RecD (5'-to-3' helicase), and RecC. Here we used a single-molecule tethered particle motion technique to directly monitor the translocation of RecBCD along chi-contained DNA molecules. Using bead-labeled enzymes, we monitored the RecBCD translocation along individual DNA by measuring the gradual decrease in the bead Brownian motion as the enzyme moves along the DNA towards the surface. DNA substrates were designed that RecBCD would encounter a single-stranded DNA gap after the recognition of the chi sequence. While translocating along chi-free DNA substrates, the time traces showed no apparent pause, which neither 3'-to-5' nor 5'-to-3' ssDNA gap influents the movement of the enzyme1. However, over 50% of RecBCD enzymes failed to pass through the 5'-to-3' ssDNA gap after translocating over chi-containing duplex DNA. Considering RecD as a major 5'-to-3' ssDNA translocase in the RecBCD complex, our observation is consistent with the model that the conformation change occurs after chi recognition and RecD is disengaged from the 5'-ssDNA.(1) Chung, C.; Li, H. W. J. Am. Chem. Soc. 2013, 135, 8920.
-
direct observation of RecBCD helicase as single stranded dna translocases
Journal of the American Chemical Society, 2013Co-Authors: Cinya Chung, Hungwen LiAbstract:The heterotrimeric Escherichia coli RecBCD enzyme comprises two helicase motors with different polarities: RecB (3′-to-5′) and RecD (5′-to-3′). This superfamily I helicase is responsible for initiating DNA double-strand-break (DSB) repair in the homologous recombination pathway. We used single-molecule tethered particle motion (TPM) experiments to visualize the RecBCD helicase translocation over long single-stranded (ss) DNA (>200 nt) with no apparent secondary structure. The bead-labeled RecBCD helicases were found to bind to the surface-immobilized blunt-end DNA, and translocate along the DNA substrates containing an ssDNA gap, resulting in a gradual decrease in the bead Brownian motion. Successful observation of RecBCD translocation over a long gap in either 3′-to-5′ or 5′-to-3′ ssDNA direction indicates that RecBCD helicase possesses ssDNA translocase activities in both polarities. Most RecBCD active tethers showed full translocation across the ssDNA to the dsDNA region, with about 19% of enzymes diss...
-
Direct Observation of RecBCD Helicase as ssDNA Translocases
Biophysical Journal, 2013Co-Authors: Cinya Chung, Hungwen LiAbstract:The E. coli RecBCD is a heterotrimeric enzyme composed of two helicase motors with different polarities: RecB (3’-to-5’) and RecD (5’-to-3’). This Superfamily I helicase is responsible for the initiation of DNA double-strand-break (DSB) repair in the homologous recombination pathway. We used single-molecule tethered particle motion (TPM) experiments to visualize the RecBCD helicase translocation over long single-stranded (ss) DNA (> 200 nt) without secondary structure. The bead-labeled RecBCD helicases were found to bind to the surface-immobilized blunt duplex DNA, and translocate along the duplex/single-stranded/duplex DNA substrate, resulting in a gradual decrease in the bead Brownian motion. Successful observation of RecBCD translocation over long ssDNA gap in either 3’-to-5’ or 5’-to-3’ direction indicates that both RecB and RecD are ssDNA translocases. We also applied continuous force (∼ 0.2 pN) to stretch DNA substrates and to remove any potentially transient looped ssDNA structure, and observed continuous translocation of RecBCD. It confirms the ssDNA translocase activities of RecBCD helicase. About 78 % of active tethers showed full translocation across the ssDNA to the dsDNA region, and the other 22 % enzymes dissociated from the ss/dsDNA junction after translocating across the ssDNA region. We also prepared a double-gapped substrate containing two regions of ssDNA with opposite polarities (5’-to-3’ and 3’-to-5’) intermitted by duplex DNA. RecBCD was able to translocate across both ssDNA regions in either ssDNA orientation orders, with 20 - 40 % of tethers dissociating while entering the second ssDNA region. These results suggest a mechanism that RecBCD is able to switch motors and rethread into the other strand after translocating along an ssDNA gapped region.
-
using a single molecule method to visualize RecBCD helicase translocation along single stranded dna
Biophysical Journal, 2012Co-Authors: Cinya ChungAbstract:The E. coli RecBCD helicase initiates the repair of double strand DNA break in the homologous recombination pathway. RecBCD is a heterotrimeric enzyme composed of two helicase motors with different polarities: RecB is a 3’-to- 5’ helicase and RecD is a 5’-to-3’ helicase. How RecBCD unwinds and translocates along duplex DNA is not clearly defined. Here we used a single-molecule tethered particle motion (TPM) experiment to visualize the RecBCD helicase translocation over long distance single-stranded (ss) DNA. We first prepared DNA substrates containing a > 200 nt long, unstructured ssDNA gap flanked by double-stranded DNA for RecBCD loading. In the TPM experiments, the bead-labeled, biotinylated RecBCD helicases are found to recognize and bind to the blunt, double-stranded DNA end, and successfully translocate along the duplex/single-stranded/duplex DNA substrate, resulting in a gradual decrease in the bead Brownian motion amplitude. Successful observation of RecBCD translocation over long ssDNA gap in either 3’-to- 5’ or 5’-to-3’ direction indicates that wild-type RecBCD functions an ssDNA translocase.
Timothy M Lohman - One of the best experts on this subject based on the ideXlab platform.
-
processive dna unwinding by RecBCD helicase in the absence of canonical motor translocation
Journal of Molecular Biology, 2016Co-Authors: Michael J. Simon, Joshua E. Sokoloski, Elizabeth Weiland, Timothy M LohmanAbstract:Abstract Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3′ to 5′ translocase, and RecD, a 5′ to 3′ translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair melting. However, some biochemical evidence suggests that DNA melting of multiple base pairs may occur separately from single-stranded DNA translocation. To test this hypothesis, we designed DNA substrates containing reverse backbone polarity linkages that prevent ssDNA translocation of the canonical RecB and RecD motors. Surprisingly, we find that RecBCD can processively unwind DNA for at least 80 bp beyond the reverse polarity linkages. This ability requires an ATPase active RecB motor, the RecB “arm” domain, and also the RecB nuclease domain, but not its nuclease activity. These results indicate that RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by the canonical motors and that the nuclease domain regulates the helicase activity of RecBCD.
-
Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation
Biophysical Journal, 2016Co-Authors: Michael J. Simon, Joshua E. Sokoloski, Elizabeth Weiland, Timothy M LohmanAbstract:E. coli RecBCD is a DNA helicase that functions in repair of double-stranded DNA breaks. RecBCD possesses two SF1 ATPase motors (RecB, a 3' to 5' translocase, and RecD, a 5' to 3' translocase). Structure-based models for DNA unwinding by RecBCD propose that RecB and RecD motor translocation is tightly coupled to base pair (bp) melting in that 1 bp is melted as the motors translocate along each single strand by 1 nucleotide for each ATP hydrolyzed. However, there is biochemical evidence to suggest that DNA melting of multiple bp may occur separately from ssDNA translocation and that these two activities may be separable. To test this hypothesis, we designed DNA substrates containing reversals of the phosphodiester backbone polarity (3'-3' and 5'-5' RP linkages) at the same point within the complementary DNA strands that prevent translocation of the canonical RecB and RecD motors. Even though the canonical motors cannot translocate, we observe processive DNA unwinding of at least 80 bp beyond the RP linkages by both RecBCD and RecBC, hence DNA unwinding and ssDNA translocation are separable. This ability is dependent on the previously identified secondary translocase activity residing within RecBC, but does not require RecD. Surprisingly, this ability also requires the nuclease domain of RecB, but not its nuclease activity. These results suggest that RecBCD may couple a double stranded DNA translocase activity to DNA melting and that the nuclease domain plays either an active role in or regulates the helicase activity of RecBCD (supported by NIH GM045948 to TML).
-
asymmetric regulation of bipolar single stranded dna translocation by the two motors within escherichia coli RecBCD helicase
Journal of Biological Chemistry, 2013Co-Authors: Colin G Wu, Elizabeth Weiland, Timothy M LohmanAbstract:Abstract Repair of double stranded DNA breaks in E. coli is initiated by the RecBCD helicase that possesses two superfamily-1 motors: RecB (3″ to 5″ translocase) and RecD (5″ to 3″ translocase) that operate on the complementary DNA strands to unwind duplex DNA. However, it is not known whether the RecB and RecD motors act independently or are functionally coupled. Here we show by directly monitoring ATP-driven ssDNA translocation of RecBCD that the 5″ to 3″ rate is always faster than the 3″ to 5″ rate on DNA without a Chi site and that the translocation rates are coupled asymmetrically. That is, RecB regulates both 3″ to 5″ and 5″ to 3″ translocation, whereas RecD only regulates 5″ to 3″ translocation. We show that the recently identified RecBC secondary translocase activity functions within RecBCD and that this contributes to the coupling. This coupling has implications for how RecBCD activity is regulated after it recognizes a Chi sequence during DNA unwinding.
-
influence of dna end structure on the mechanism of initiation of dna unwinding by the escherichia coli RecBCD and recbc helicases
Journal of Molecular Biology, 2008Co-Authors: Colin G Wu, Timothy M LohmanAbstract:Abstract Escherichia coli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3′-to-5′ translocase, and RecD, a 5′-to-3′ translocase) that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n -step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can “melt out” ∼ 6 bp upon binding to the end of a blunt-ended DNA duplex in a Mg 2+ -dependent but ATP-independent reaction, we investigated the effects of noncomplementary single-stranded (ss) DNA tails [3′-(dT) 6 and 5′-(dT) 6 or 5′-(dT) 10 ] on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3′-(dT) 6 and 5′-(dT) 6 noncomplementary ssDNA tails is well described by a sequential n -step mechanism with the same unwinding rate ( mk U = 774 ± 16 bp s − 1 ) and kinetic step size ( m = 3.3 ± 1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding ( k C = 45 ± 2 s − 1 ). However, when the noncomplementary 5′ ssDNA tail is extended to 10 nt [5′-(dT) 10 and 3′-(dT) 6 ], the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate ( mk U = 538 ± 24 bp s − 1 ) is observed with a similar kinetic step size ( m = 3.9 ± 0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing noncomplementary twin 5′-(dT) 6 and 3′-(dT) 6 tails, and unwinding can be described by a simple uniform n -step sequential scheme, without the need for the additional k C initiation steps, with a similar kinetic step size ( m = 4.4 ± 1.7 bp) and unwinding rate ( mk obs = 396 ± 15 bp s − 1 ). These results suggest that the additional kinetic steps with rate constant k C required for RecBCD to initiate unwinding of blunt-ended and twin (dT) 6 -tailed DNA reflect processes needed to engage the RecD motor with the 5′ ssDNA.
-
probing 3 ssdna loop formation in e coli RecBCD recbc dna complexes using non natural dna a model for chi recognition complexes
Journal of Molecular Biology, 2006Co-Authors: Jason C Wong, Rachel L Rice, Nathan A Baker, Tao Ju, Timothy M LohmanAbstract:Abstract The equilibrium binding of Escherichia coli RecBC and RecBCD helicases to duplex DNA ends containing varying lengths of polyethylene glycol (PEG) spacers within pre-formed 3′-single-stranded (ss) DNA ((dT)n) tails was studied. These studies were designed to test a previous proposal that the 3′-(dT)n tail can be looped out upon binding RecBC and RecBCD for 3′-ssDNA tails with n ≥ 6 nucleotides. Equilibrium binding of protein to unlabeled DNA substrates with ends containing PEG-substituted 3′-ssDNA tails was examined by competition with a Cy3-labeled reference DNA which undergoes a Cy3 fluorescence enhancement upon protein binding. We find that the binding affinities of both RecBC and RecBCD for a DNA end are unaffected upon substituting PEG for the ssDNA between the sixth and the final two nucleotides of the 3′-(dT)n tail. However, placing PEG at the end of the 3′-(dT)n tail increases the binding affinities to their maximum values (i.e. the same as binding constants for RecBC or RecBCD to a DNA end with only a 3′-(dT)6 tail). Equilibrium binding studies of a RecBC mutant containing a nuclease domain deletion, RecBΔnucC, suggest that looping of the 3′-tail (when n ≥ 6 nucleotides) occurs even in the absence of the RecB nuclease domain, although the nuclease domain stabilizes such loop formation. Computer modeling of the RecBCD–DNA complexes suggests that the loop in the 3′-ssDNA tail may form at the RecB/RecC interface. Based on these results we suggest a model for how a loop in the 3′-ssDNA tail might form upon encounter of a “Chi” recognition sequence during unwinding of DNA by the RecBCD helicase.
