ATP Hydrolysis in the RecA–DNA Filament Promotes Structural Changes at the Protein–DNA Interface

Reymer, Anna; Babik, Sándor; Takahashi, Masayuki; Nordén, Bengt; Beke‐Somfai, Tamás

doi:10.1021/acs.biochem.5b00614

Cited by 10 publications

(8 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…DSB (double-strand breakage) caused by Cas9 should be precisely repaired through homology directed recombination (HDR), which is exerted by the ATP-dependent DNA repair system. ATP plays important and essential roles in HDR, as it is associated with recombinase (RecA/Rad51) filaments on DNA, and its hydrolysis is essential for the dynamic interaction between RecA-ssDNA (Reymer et al, 2015), RecA sliding along DNA (Kim et al, 2017), and conformational change during Rad51 filament disassembly (Brouwer et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Multi-Layer Controls of Cas9 Activity Coupled With ATP Synthase Over-Expression for Efficient Genome Editing in Streptomyces

Wang

Zhao

Liu

et al. 2019

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

Efficient genome editing is a prerequisite of genetic engineering in synthetic biology, which has been recently achieved by the powerful CRISPR/Cas9 system. However, the toxicity of Cas9, due to its abundant intracellular expression, has impeded its extensive applications. Here we constructed a genetic cassette with triple controls of Cas9 activities at transcriptional, translational and protein levels, together with over-expression of the ATP synthase β-subunit AtpD, for the efficient genome editing in Streptomyces. By deletion of actII-ORF4 in Streptomyces coelicolor as a model, we found that constitutive expression of cas9 had about 90% editing efficiency but dramatically reduced transformation efficiency by 900-fold. However, triple controls of Cas9 under non-induction conditions to reduce its activity increased transformation efficiency over 250-fold, and had about 10% editing efficiency if combined with atpD overexpression. Overall, our strategy accounts for about 30-fold increased possibility for successful genome editing under the non-induction condition. In addition, about 80% editing efficiency was observed at the actII-ORF4 locus after simultaneous induction with thiostrepton, theophylline and blue light for Cas9 activity reconstitution. This improved straightforward efficient genome editing was also confirmed in another locus redD. Thus, we developed a new strategy for efficient genome editing, and it could be readily and widely adaptable to other Streptomyces species to improve genetic manipulation for rapid strain engineering in Streptomyces synthetic biology, due to the highly conserved genetic cassettes in this genus.

show abstract

Section: Introductionmentioning

confidence: 99%

Multi-Layer Controls of Cas9 Activity Coupled With ATP Synthase Over-Expression for Efficient Genome Editing in Streptomyces

Wang

Zhao

Liu

et al. 2019

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

show abstract

“…Notably, the ATP cofactor is buried in the interface between adjacent monomers in the extended form, while it is accessible to the solvent in the compressed form. At the local level, quantum chemical studies indicate a reorganization of the hydrogen-bond network at the protein–DNA interface (25).…”

Section: Introductionmentioning

confidence: 99%

Weaving DNA strands: structural insight on ATP hydrolysis in RecA-induced homologous recombination

Boyer

Danilowicz

Prentiss

et al. 2019

Nucleic Acids Research

View full text Add to dashboard Cite

Homologous recombination is a fundamental process in all living organisms that allows the faithful repair of DNA double strand breaks, through the exchange of DNA strands between homologous regions of the genome. Results of three decades of investigation and recent fruitful observations have unveiled key elements of the reaction mechanism, which proceeds along nucleofilaments of recombinase proteins of the RecA family. Yet, one essential aspect of homologous recombination has largely been overlooked when deciphering the mechanism: while ATP is hydrolyzed in large quantity during the process, how exactly hydrolysis influences the DNA strand exchange reaction at the structural level remains to be elucidated. In this study, we build on a previous geometrical approach that studied the RecA filament variability without bound DNA to examine the putative implication of ATP hydrolysis on the structure, position, and interactions of up to three DNA strands within the RecA nucleofilament. Simulation results on modeled intermediates in the ATP cycle bring important clues about how local distortions in the DNA strand geometries resulting from ATP hydrolysis can aid sequence recognition by promoting local melting of already formed DNA heteroduplex and transient reverse strand exchange in a weaving type of mechanism.

show abstract

“…Hydrolysis of ATP provokes large‐scale conformational changes resulting in compression of the stretched active RecA filament into a state, generally referred as inactive. ATP hydrolysis modulates RecA‐DNA interactions through the network of hydrogen bonds connecting ATP‐binding and DNA‐binding sites [9,68]. In this work, using a single‐molecule approach, we were able to identify two distinct compressed states of the RecA‐ssDNA nucleoprotein filament, which we refer to as ADP and Apo states.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

View full text Add to dashboard Cite

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.

show abstract

ATP Hydrolysis in the RecA–DNA Filament Promotes Structural Changes at the Protein–DNA Interface

Cited by 10 publications

References 23 publications

Multi-Layer Controls of Cas9 Activity Coupled With ATP Synthase Over-Expression for Efficient Genome Editing in Streptomyces

Multi-Layer Controls of Cas9 Activity Coupled With ATP Synthase Over-Expression for Efficient Genome Editing in Streptomyces

Weaving DNA strands: structural insight on ATP hydrolysis in RecA-induced homologous recombination

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

Contact Info

Product

Resources

About