Faculty Opinions recommendation of Mechanics and Single-Molecule Interrogation of DNA Recombination.

2018 ◽  
Author(s):  
Roland Kanaar
Keyword(s):  
Single Molecule ◽  
Dna Recombination

scholarly journals Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination

2017 ◽  
Vol 114 (4) ◽  
pp. E466-E475 ◽  
Author(s):  
Gábor M. Harami ◽  
Yeonee Seol ◽  
Junghoon In ◽  
Veronika Ferencziová ◽  
Máté Martina ◽  
...  
Keyword(s):  
Single Molecule ◽  
Domain Architecture ◽  
Dna Recombination ◽  
Recq Helicase ◽  
Dna Structures ◽  
Recq Helicases ◽  
Differential Recognition ◽  
D Loop ◽  
Mechanistic Basis

Cells must continuously repair inevitable DNA damage while avoiding the deleterious consequences of imprecise repair. Distinction between legitimate and illegitimate repair processes is thought to be achieved in part through differential recognition and processing of specific noncanonical DNA structures, although the mechanistic basis of discrimination remains poorly defined. Here, we show thatEscherichia coliRecQ, a central DNA recombination and repair enzyme, exhibits differential processing of DNA substrates based on their geometry and structure. Through single-molecule and ensemble biophysical experiments, we elucidate how the conserved domain architecture of RecQ supports geometry-dependent shuttling and directed processing of recombination-intermediate [displacement loop (D-loop)] substrates. Our study shows that these activities together suppress illegitimate recombination in vivo, whereas unregulated duplex unwinding is detrimental for recombination precision. Based on these results, we propose a mechanism through which RecQ helicases achieve recombination precision and efficiency.

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 Single-Molecule Fluorescence Studies of Site-Specific DNA Recombination

2012 ◽  
Vol 102 (3) ◽  
pp. 283a
Author(s):  
Justin Pinkney ◽  
Pawel Zawadzki ◽  
David J. Sherratt ◽  
Achillefs N. Kapanidis
Keyword(s):  
Single Molecule ◽  
Dna Recombination ◽  
Single Molecule Fluorescence ◽  
Site Specific ◽  
Fluorescence Studies

scholarly journals Single-molecule Study of Site-specific DNA Recombination by γδ Resolvase

2009 ◽  
Vol 96 (3) ◽  
pp. 59a
Author(s):  
Mingxuan Sun ◽  
Hua Bai ◽  
Nigel D. Grindley ◽  
John F. Marko
Keyword(s):  
Single Molecule ◽  
Dna Recombination ◽  
Site Specific

scholarly journals Reappraising the human mitochondrial DNA recombination dogma

2018 ◽  
Author(s):  
Simόn Perera ◽  
Amanda Ramos ◽  
Luis Alvarez ◽  
Débora Jurado ◽  
Maria Guardiola ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Single Molecule ◽  
Dna Recombination ◽  
Recent Common Ancestor ◽  
Human Variation ◽  
Substantial Impact ◽  
Human Mitochondrial Dna ◽  
Mtdna Recombination ◽  
Most Recent Common Ancestor ◽  
Frequent Event

AbstractWith the “mitochondrial Eve” theory proposed by Rebecca Cann in the eighties, human mitochondrial DNA (mtDNA) has been used as a tool in studying human variation and evolution. Although the existence of recombination in human mtDNA has been previously advocated, studies dealing with human variation and evolution have assumed that human mtDNA does not recombine and should be considered as pathological or very infrequent. Using both direct and indirect approaches, we provide consistent evidence of mtDNA recombination in humans. We applied the single molecule PCR procedure to directly test for recombination in multiheteroplasmic individuals without any overt pathology. Moreover, we searched for past recombination events in the whole mitochondrial genomes of more than 15,000 individuals. Results from our study update and expand both the seminal indirect findings and the scarce direct evidence observed to date, paving the way for the definitive rejection of the non-recombination dogma for human mtDNA. Acknowledgment of recombination as a frequent event in mtDNA will require the description of the population recombination rate(s) and to apply it to past and future studies involving mtDNA. MtDNA recombination affects our knowledge of human evolutionary history, regarding haplogroup divergence times, as well as the time to the mitochondrial most recent common ancestor. Finally, mtDNA recombination will have a substantial impact on our understanding of the etiology and transmission of mitochondrial diseases.

Mechanics and Single-Molecule Interrogation of DNA Recombination

2016 ◽  
Vol 85 (1) ◽  
pp. 193-226 ◽  
Author(s):  
Jason C. Bell ◽  
Stephen C. Kowalczykowski
Keyword(s):  
Single Molecule ◽  
Dna Recombination

scholarly journals Single-Molecule Microscopy Meets Molecular Dynamics Simulations for Characterizing the Molecular Action of Proteins on DNA and in Liquid Condensates

2021 ◽  
Vol 8 ◽  
Author(s):  
Kiyoto Kamagata
Keyword(s):  
Molecular Dynamics ◽  
Dna Binding ◽  
Molecular Dynamics Simulations ◽  
Single Molecule ◽  
Binding Proteins ◽  
Dynamic Properties ◽  
Dna Recombination ◽  
Dna Binding Proteins ◽  
Molecular Action ◽  
Dynamics Simulations

DNA-binding proteins trigger various cellular functions and determine cellular fate. Before performing functions such as transcription, DNA repair, and DNA recombination, DNA-binding proteins need to search for and bind to their target sites in genomic DNA. Under evolutionary pressure, DNA-binding proteins have gained accurate and rapid target search and binding strategies that combine three-dimensional search in solution, one-dimensional sliding along DNA, hopping and jumping on DNA, and intersegmental transfer between two DNA molecules. These mechanisms can be achieved by the unique structural and dynamic properties of these proteins. Single-molecule fluorescence microscopy and molecular dynamics simulations have characterized the molecular actions of DNA-binding proteins in detail. Furthermore, these methodologies have begun to characterize liquid condensates induced by liquid-liquid phase separation, e.g., molecular principles of uptake and dynamics in droplets. This review discusses the molecular action of DNA-binding proteins on DNA and in liquid condensate based on the latest studies that mainly focused on the model protein p53.

The method of replication affects metal film granularity and resolution

1989 ◽  
Vol 47 ◽  
pp. 708-709
Author(s):  
George C. Ruben
Keyword(s):  
Electron Beam ◽  
High Resolution ◽  
Film Thickness ◽  
Single Molecule ◽  
Metal Film ◽  
Vertical Surface ◽  
High Resolution Imaging ◽  
Controllable Factors ◽  
Resolution Imaging

Single molecule resolution in electron beam sensitive, uncoated, noncrystalline materials has been impossible except in thin Pt-C replicas ≤ 150Å) which are resistant to the electron beam destruction. Previously the granularity of metal film replicas limited their resolution to ≥ 20Å. This paper demonstrates that Pt-C film granularity and resolution are a function of the method of replication and other controllable factors. Low angle 20° rotary , 45° unidirectional and vertical 9.7±1 Å Pt-C films deposited on mica under the same conditions were compared in Fig. 1. Vertical replication had a 5A granularity (Fig. 1c), the highest resolution (table), and coated the whole surface. 45° replication had a 9Å granulartiy (Fig. 1b), a slightly poorer resolution (table) and did not coat the whole surface. 20° rotary replication was unsuitable for high resolution imaging with 20-25Å granularity (Fig. 1a) and resolution 2-3 times poorer (table). Resolution is defined here as the greatest distance for which the metal coat on two opposing faces just grow together, that is, two times the apparent film thickness on a single vertical surface.

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