scholarly journals Single-Molecule Studies of the ssDNA Binding Activity of E. Coli MutL

2010 ◽  
Vol 98 (3) ◽  
pp. 64a-65a
Author(s):  
Jonghyun Park ◽  
Yong-Moon Jeon ◽  
Daekil In ◽  
Seong-Dal Heo ◽  
Changill Ban ◽  
...  
Keyword(s):  
Single Molecule ◽  
Binding Activity ◽  
Ssdna Binding ◽  
E Coli ◽  
Single Molecule Studies

scholarly journals Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Thomas J Etheridge ◽  
Desiree Villahermosa ◽  
Eduard Campillo-Funollet ◽  
Alex David Herbert ◽  
Anja Irmisch ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Single Molecule ◽  
Chromosomal Rearrangements ◽  
Binding Activity ◽  
Single Molecule Tracking ◽  
Ssdna Binding ◽  
Dna Binding Activity ◽  
Gross Chromosomal Rearrangements ◽  
Genes Encoding

The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits, we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the single-stranded DNA (ssDNA) binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.

scholarly journals Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates

2020 ◽  
Author(s):  
Chaoyou Xue ◽  
Lucia Molnarova ◽  
Justin B Steinfeld ◽  
Weixing Zhao ◽  
Chujian Ma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Protein A ◽  
Dna Recombination ◽  
Binding Activity ◽  
Negative Regulator ◽  
Genome Maintenance ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Translocation Velocity

Abstract RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

scholarly journals The HRDC domain oppositely modulates the unwinding activity of E. coli RecQ helicase on duplex DNA and G-quadruplex

2020 ◽  
Vol 295 (51) ◽  
pp. 17646-17658
Author(s):  
Fang-Yuan Teng ◽  
Ting-Ting Wang ◽  
Hai-Lei Guo ◽  
Ben-Ge Xin ◽  
Bo Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Stability ◽  
Premature Aging ◽  
Binding Modes ◽  
Ssdna Binding ◽  
Recq Helicases ◽  
E Coli ◽  
Single Molecule Fret ◽  
G4 Dna ◽  
Long Time

RecQ family helicases are highly conserved from bacteria to humans and have essential roles in maintaining genome stability. Mutations in three human RecQ helicases cause severe diseases with the main features of premature aging and cancer predisposition. Most RecQ helicases shared a conserved domain arrangement which comprises a helicase core, an RecQ C-terminal domain, and an auxiliary element helicase and RNaseD C-terminal (HRDC) domain, the functions of which are poorly understood. In this study, we systematically characterized the roles of the HRDC domain in E. coli RecQ in various DNA transactions by single-molecule FRET. We found that RecQ repetitively unwinds the 3′-partial duplex and fork DNA with a moderate processivity and periodically patrols on the ssDNA in the 5′-partial duplex by translocation. The HRDC domain significantly suppresses RecQ activities in the above transactions. In sharp contrast, the HRDC domain is essential for the deep and long-time unfolding of the G4 DNA structure by RecQ. Based on the observations that the HRDC domain dynamically switches between RecA core- and ssDNA-binding modes after RecQ association with DNA, we proposed a model to explain the modulation mechanism of the HRDC domain. Our findings not only provide new insights into the activities of RecQ on different substrates but also highlight the novel functions of the HRDC domain in DNA metabolisms.

scholarly journals Facilitated Dissociation of Nucleoid Associated Proteins from DNA in the Bacterial Confinement

2021 ◽  
Author(s):  
Zafer Koşar ◽  
A. Göktuĝ Attar ◽  
Aykut Erbaş
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Experimental Studies ◽  
Dual Purpose ◽  
E Coli ◽  
Protein Dna Interactions ◽  
Factor For Inversion Stimulation ◽  
Associated Proteins ◽  
Order Of Magnitude ◽  
Single Molecule Studies

Transcription machinery ultimately depends on the temporal formation of protein-DNA complexes. Recent experimental studies demonstrate that residence time (i.e., inverse off-rate) of a transcription factor protein can be a contributor to the functional diversity of the protein. In the meantime, single-molecule experiments showed that the off-rates of a wide array of DNA-binding proteins accelerate as the bulk concentration of the protein increases via a concentration-dependent mechanism (i.e., facilitated dissociation, FD). In this study, inspired by the previous single-molecule studies on the factor for inversion stimulation (Fis) protein of E. coli, which is a dual-purpose protein with a diverse functionality, we model the unbinding of Fis from specific bindings sites along a high-molecular-weight circular DNA in a cylindrical structure mimicking the cellular confinement of chromosome. Our simulations show that FD of Fis can well occur in confinement at physiological concentrations. Particularly, when nutrient-rich conditions are emulated with Fis concentrations around micromolar levels, the off-rates increase one order of magnitude compared to the lower Fis levels. However, Fis significantly changes the chromosome structure at higher concentrations by forming dense protein clusters bridging specific sites and juxtaposing remote DNA segments. As a result, at the physiologically observed maximum levels of Fis, the off-rates significantly slow down. Overall, our results indicate that cellular-concentration levels of a structural DNA-binding protein is intermingled with the genome architecture and DNA residence times, thereby providing a basis for understanding the complex effects of dynamic protein-DNA interactions on gene regulation.

scholarly journals Single-molecule live cell imaging of the Smc5/6 DNA repair complex

2020 ◽  
Author(s):  
Thomas J. Etheridge ◽  
Desiree Villahermosa ◽  
Anja Irmisch ◽  
Adam T. Watson ◽  
Alex Herbert ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Single Molecule ◽  
Chromosomal Rearrangements ◽  
Binding Activity ◽  
Single Molecule Tracking ◽  
Ssdna Binding ◽  
Dna Binding Activity ◽  
Double Stranded Dna ◽  
Gross Chromosomal Rearrangements ◽  
Genes Encoding

AbstractThe Smc5/6 complex is involved in various DNA transactions and is best known for ensuring the fidelity of homologous recombination. We exploit single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association. We show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits we show that the Nse3 double-stranded DNA binding activity and the two arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the ssDNA binding activity at the hinge region does not prevent chromatin association. However, unlike a mutant attenuating chromatin association, a mutant that disrupts ssDNA binding results in highly elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.

scholarly journals Single-molecule studies contrast ordered DNA replication with stochastic translesion synthesis

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Gengjing Zhao ◽  
Emma S Gleave ◽  
Meindert Hugo Lamers
Keyword(s):  
Dna Adducts ◽  
Single Molecule ◽  
Uv Light ◽  
Dna Polymerases ◽  
Stable Complex ◽  
High Fidelity ◽  
Single Molecule Spectroscopy ◽  
E Coli ◽  
Single Molecule Studies ◽  
Translesion Polymerases

High fidelity replicative DNA polymerases are unable to synthesize past DNA adducts that result from diverse chemicals, reactive oxygen species or UV light. To bypass these replication blocks, cells utilize specialized translesion DNA polymerases that are intrinsically error prone and associated with mutagenesis, drug resistance, and cancer. How untimely access of translesion polymerases to DNA is prevented is poorly understood. Here we use co-localization single-molecule spectroscopy (CoSMoS) to follow the exchange of the E. coli replicative DNA polymerase Pol IIIcore with the translesion polymerases Pol II and Pol IV. We find that in contrast to the toolbelt model, the replicative and translesion polymerases do not form a stable complex on one clamp but alternate their binding. Furthermore, while the loading of clamp and Pol IIIcore is highly organized, the exchange with the translesion polymerases is stochastic and is not determined by lesion-recognition but instead a concentration-dependent competition between the polymerases.

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