Kinetics of the ATP and dATP-mediated formation of a functionally-active RecA-ssDNA complex

Nayak, Sunil; Bryant, Floyd R.

doi:10.1016/j.bbrc.2015.06.097

Cited by 10 publications

(13 citation statements)

References 18 publications

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“…We thus measured the extent of ATP/dATP hydrolysis by the npRecA dimer compared with the RecA controls, in the presence of sodium acetate (1.5 M) instead of DNA. It has been reported that dATP is a more effective nucleotide cofactor than ATP for the molecular activities of E. coli RecA ( 44 , 45 ). In agreement with these reports, we observed that more dATP was hydrolyzed by RecA-wt than ATP in the presence of sodium acetate (Figure 4A and B ).…”

Section: Resultsmentioning

confidence: 99%

Nonfilament-forming RecA dimer catalyzes homologous joint formation

Shinohara

Arai

Iikura

et al. 2018

Nucleic Acids Research

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Homologous recombination is essential to genome maintenance, and also to genome diversification. In virtually all organisms, homologous recombination depends on the RecA/Rad51-family recombinases, which catalyze ATP-dependent formation of homologous joints—critical intermediates in homologous recombination. RecA/Rad51 binds first to single-stranded (ss) DNA at a damaged site to form a spiral nucleoprotein filament, after which double-stranded (ds) DNA interacts with the filament to search for sequence homology and to form consecutive base pairs with ssDNA (‘pairing’). How sequence homology is recognized and what exact role filament formation plays remain unknown. We addressed the question of whether filament formation is a prerequisite for homologous joint formation. To this end we constructed a nonpolymerizing (np) head-to-tail-fused RecA dimer (npRecA dimer) and an npRecA monomer. The npRecA dimer bound to ssDNA, but did not form continuous filaments upon binding to DNA; it formed beads-on-string structures exclusively. Although its efficiency was lower, the npRecA dimer catalyzed the formation of D-loops (a type of homologous joint), whereas the npRecA monomer was completely defective. Thus, filament formation contributes to efficiency, but is not essential to sequence-homology recognition and pairing, for which a head-to-tail dimer form of RecA protomer is required and sufficient.

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Section: Resultsmentioning

confidence: 99%

Nonfilament-forming RecA dimer catalyzes homologous joint formation

Shinohara

Arai

Iikura

et al. 2018

Nucleic Acids Research

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“…In contrast, hydrolysis is significant when either ATP or dATP is the cofactor. Filaments prepared in ATPγS or dATP show there is a higher affinity of RecA for ssDNA ( 35 ), in comparison with ATP ( 35 ), and the time course of filament formation is the same in dATP as in ATPγS ( 36 ). Thus, if an effect is present when dATP is the cofactor, but absent with ATPγS, then the effect may be due to hydrolysis.…”

Section: Resultsmentioning

confidence: 99%

ATP hydrolysis provides functions that promote rejection of pairings between different copies of long repeated sequences

Danilowicz

Hermans

Coljee

et al. 2017

Nucleic Acids Research

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During DNA recombination and repair, RecA family proteins must promote rapid joining of homologous DNA. Repeated sequences with >100 base pair lengths occupy more than 1% of bacterial genomes; however, commitment to strand exchange was believed to occur after testing ∼20–30 bp. If that were true, pairings between different copies of long repeated sequences would usually become irreversible. Our experiments reveal that in the presence of ATP hydrolysis even 75 bp sequence-matched strand exchange products remain quite reversible. Experiments also indicate that when ATP hydrolysis is present, flanking heterologous dsDNA regions increase the reversibility of sequence matched strand exchange products with lengths up to ∼75 bp. Results of molecular dynamics simulations provide insight into how ATP hydrolysis destabilizes strand exchange products. These results inspired a model that shows how pairings between long repeated sequences could be efficiently rejected even though most homologous pairings form irreversible products.

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“…The nucleoprotein filament thus formed is called the presynaptic filament. In this first conformation, in which the protein is inactive because of the absence of its cofactors, RecA interacts with five nucleotides (23,24).…”

Section: Formation Of the Presynaptic Filamentmentioning

confidence: 99%

RecA and DNA recombination: a review of molecular mechanisms

Val

Nasser

Abaibou

et al. 2019

Biochemical Society Transactions

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Recombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson–Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg2+, two cofactors required for RecA activity. Then, the complex starts a homology search by interacting with and stretching dsDNA. Thanks to supercoiling, intersegment sampling and RecA clustering, a genome-wide homology search takes place at a relevant metabolic timescale. When a region of homology 8–20 base pairs in length is found and stabilized, DNA strand exchange proceeds, forming a heteroduplex complex that is resolved through a combination of DNA synthesis, ligation and resolution. RecA activities can take place without ATP hydrolysis, but this latter activity is necessary to improve and accelerate the process. Protein flexibility and monomer–monomer interactions are fundamental for RecA activity, which functions cooperatively. A structure/function relationship analysis suggests that the recombinogenic activity can be improved and that recombinases have an inherently large recombination potential. Understanding this relationship is essential for designing RecA derivatives with enhanced activity for biotechnology applications. For example, this protein is a major actor in the recombinase polymerase isothermal amplification (RPA) used in point-of-care diagnostics.

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Kinetics of the ATP and dATP-mediated formation of a functionally-active RecA-ssDNA complex

Cited by 10 publications

References 18 publications

Nonfilament-forming RecA dimer catalyzes homologous joint formation

Nonfilament-forming RecA dimer catalyzes homologous joint formation

ATP hydrolysis provides functions that promote rejection of pairings between different copies of long repeated sequences

RecA and DNA recombination: a review of molecular mechanisms

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