Deinococcus radiodurans RecA nucleoprotein filaments characterized at the single-molecule level with optical tweezers

Pobegalov, Georgii; Cherevatenko, Galina; Alekseev, Aleksandr; Sabantsev, Anton; Kovaleva, Oksana; Vedyaykin, Alexey; Morozova, Natalia; Baitin, Dmitry; Khodorkovskii, M. A.

doi:10.1016/j.bbrc.2015.09.042

Cited by 14 publications

(19 citation statements)

References 28 publications

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“…In order to examine the interaction of DrRecA with ssDNA, we initially followed the change in length of individual DNA tethers in the presence of DrRecA and ATP using an approach combining DNA manipulation by optical tweezers and a multichannel microfluidic flow system (uFlux, Lumicks) published elsewhere [ 42 , 43 , 44 , 45 ]. The formation of a nucleoprotein complex by RecA-family proteins leads to a drastic change in the mechanical properties of the DNA molecule at the core of the complex [ 22 ].…”

Section: Resultsmentioning

confidence: 99%

“…Considering that room temperature possibly is not a favorable condition for the proper assembly of DrRecA–dsDNA filaments [ 42 , 47 ], we further tested dsDNA–DrRecA interaction at a higher temperature of 37 °C and under lower Mg 2+ ions concentration (1.5 mM MgCl 2 ). Under these conditions, the DrRecA polymerization was less constrained and proceeded under a lower applied tension of 20 pN ( Figure 3 C).…”

Section: Resultsmentioning

confidence: 99%

“…Single-molecule insights into dsDNA binding by DrRecA provided an important observation of the faster nucleation and slower elongation of DrRecA compared to EcRecA [ 41 ]. Furthermore, direct comparison of the mechanical properties of the nucleoprotein filaments formed on dsDNA revealed that DrRecA filaments are shorter and more flexible, which is a feature that might support the efficient repair of numerous concurrent DNA double-strand breaks [ 42 ]. However, little is known about the conformational dynamics of the DrRecA nucleoprotein filaments and its response to ATP hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

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Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA

Alekseev

Cherevatenko

Serdakov

et al. 2020

IJMS

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Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA

Alekseev

Cherevatenko

Serdakov

et al. 2020

IJMS

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“…Like its E. coli counterpart, drRecA forms right-handed helical filaments on DNA, hydrolyses ATP in a DNA-dependent fashion, and promotes strand exchange reactions in vitro . Importantly, however, in contrast to other known recombinase enzymes, drRecA polymerises on dsDNA [108], [109] and its polymerisation properties are significantly different from those of E. coli RecA [112] (Fig. 2 and Table 3).…”

Section: Double-strand Break Repairmentioning

confidence: 89%

“…The overall structures of dr- and E. coli RecA are similar, but changes in the relative positioning of the N- and C-terminal domains of drRecA significantly alter the pitch of the helical filament as compared to E. coli RecA, thereby producing a compressed, inactive drRecA filament. Functional insight into the key role of drRecA in the repair of hundreds of DSBs in D. radiodurans has been provided by the biochemical characterisation of drRecA initially using classical in vitro methods [108], [109], [110] and more recently using optical tweezer and single-molecule approaches [111], [112]. Like its E. coli counterpart, drRecA forms right-handed helical filaments on DNA, hydrolyses ATP in a DNA-dependent fashion, and promotes strand exchange reactions in vitro .…”

Section: Double-strand Break Repairmentioning

confidence: 99%

A Decade of Biochemical and Structural Studies of the DNA Repair Machinery of Deinococcus radiodurans: Major Findings, Functional and Mechanistic Insight and Challenges

Timmins

Moe

2016

Computational and Structural Biotechnology Journal

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The Deinococcus radiodurans bacterium is extremely resistant to ionising radiation and desiccation and can withstand a 200-fold higher radiation dose than most other bacteria with no loss of viability. The mechanisms behind this extreme resistance are not fully understood, but it is clear that several factors contribute to this phenotype. Efficient scavenging of reactive oxygen species and repair of damaged DNA are two of these. In this review, we summarise the results from a decade of structural and functional studies of the DNA repair machinery of Deinococcus radiodurans and discuss how these studies have contributed to an improved understanding of the molecular mechanisms underlying DNA repair and to the outstanding resistance of Deinococcus radiodurans to DNA damaging agents.

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Single‐molecule analysis reveals two distinct states of the compressed RecA filament on single‐stranded DNA

et al. 2020

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The RecA protein plays a key role in bacterial homologous recombination (HR) and acts through assembly of long helical filaments around single‐stranded DNA in the presence of ATP. Large‐scale conformational changes induced by ATP hydrolysis result in transitions between stretched and compressed forms of the filament. Here, using a single‐molecule approach, we show that compressed RecA nucleoprotein filaments can exist in two distinct interconvertible states depending on the presence of ADP in the monomer–monomer interface. Binding of ADP promotes cooperative conformational transitions and directly affects mechanical properties of the filament. Our findings reveal that RecA nucleoprotein filaments are able to continuously cycle between three mechanically distinct states that might have important implications for RecA‐mediated processes of HR.

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Deinococcus radiodurans RecA nucleoprotein filaments characterized at the single-molecule level with optical tweezers

Cited by 14 publications

References 28 publications

Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA

Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA

A Decade of Biochemical and Structural Studies of the DNA Repair Machinery of Deinococcus radiodurans: Major Findings, Functional and Mechanistic Insight and Challenges

Single‐molecule analysis reveals two distinct states of the compressed RecA filament on single‐stranded DNA

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