Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria

Badrinarayanan, Anjana; Le, Tung B. K.; Laub, Michael T.

doi:10.1084/jem.2129oia70

Cited by 9 publications

(31 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This search commences without major reorganization of the chromosome, excluding search strategies based on global chromosome alignment. A similar HR search time has been observed in C. crescentus, where sister pairing after a DSB occurred not in the middle but at the polar-proximal location of the uncut sister (Badrinarayanan, Le, and Laub 2015). The different pairing location in this organism may be due to the polar tethering of the C. crescentus chromosome (Mohl and Gober 1997).…”

Section: Search For a Distant Homologous Sister Locussupporting

confidence: 72%

“…The protein RecA has been implicated in the search phase of HR in a number of imaging studies (Kidane and Graumann 2005;Lesterlin et al 2014;Badrinarayanan et al 2015). To validate the timing and localizations of the events we observed with chromosomal markers, we visualised RecA dynamics under our experimental conditions.…”

Section: Reca Filaments Mediate Pairing Between Segregated Homologiesmentioning

confidence: 90%

“…Those studies showed that in E. coli the labeled RecA can form either relatively thick and stable "bundles" or short-lived spots at the site of damage (Lesterlin et al 2014;Amarh, White, and Leach 2018). In C. crescentus and Bacillus subtilis both spots and elongated RecA structures have also been observed (Badrinarayanan, Le, and Laub 2015;Kidane and Graumann 2005).…”

Section: Introductionmentioning

confidence: 99%

“…A similar mechanism for HR would be too slow, since a further complication in HR is that the diffusivity of the RecA-ssDNA filament is limited due to its large molecular mass and its tethering to the rest of the chromosome. Considering this challenge, it has been suggested that search and repair may not be possible at all between segregated sequences (Barzel & Kupiec, 2008;Weiner et al, 2009) Still it appears that long range homologous recombination can be performed within one generation time in Caulobacter crescentus (Badrinarayanan, Le, and Laub 2015) and slower in E. coli (Lesterlin et al 2014). Thus half a century after the discovery of RecA (Clark and Margulies 1965), the mechanism of search is still puzzling.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Live cell imaging reveals that RecA finds homologous DNA by reduced dimensionality search

Wiktor

Gynnå

Leroy

et al. 2020

Preprint

View full text Add to dashboard Cite

The search for a homologous template is a central and critical step in the repair of DNA breaks by homologous recombination. However, it is still unclear how the search process is carried out within a cell. Here, we image double-stranded break (DSB) repair in living E. coli growing in a microfluidic device and show that two segregated homologous sequences find each other less than 9 min after an induced DSB. To characterize the mechanisms of the search process, we use a new RecA fluorescent fusion that rapidly forms structures after DSBs. Initially, RecA forms a bright cluster at the site of the DNA damage and then extends to form a thin, flexible, and dynamic filament. Based on our observations, we propose a model where the 1D ssDNA-RecA filament stretches along the cell, and the homology search is mediated by diffusion of the repair template that can interact with any segment of the filament. This model reduces the complexity of the search from 3D to 2D and quantitatively predicts that genome-wide search for homology can be finished in minutes, in agreement with our measurements.

show abstract

Section: Search For a Distant Homologous Sister Locussupporting

confidence: 72%

Section: Reca Filaments Mediate Pairing Between Segregated Homologiesmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Live cell imaging reveals that RecA finds homologous DNA by reduced dimensionality search

Wiktor

Gynnå

Leroy

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Nucleation-competent but spreadingdefective ParB mutants formed no or very diffusive foci in vivo 19,54 . Recently, it has been observed that an artificially engineered double-strand break ~8 kb away from parS did not cause a dissolution of ParB foci in Caulobacter cells 55 . This result seemingly contradicted our findings that Caulobacter ParB spreading in vitro requires a closed DNA.…”

Section: Discussionmentioning

confidence: 99%

ParB spreading on DNA requires cytidine triphosphatein vitro

Jalal

Tran

2019

Preprint

View full text Add to dashboard Cite

In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC.Faithful chromosome segregation is essential in all domains of life if daughter cells are each to inherit the full set of genetic information. The SMC-ParAB-parS complex is widely employed for chromosome segregation in bacteria 1-13 . The centromere parS is the first DNA locus to be segregated following chromosome replication 8,9,14,15 . ParB specifically nucleates on parS before spreading outwards to the flanking DNA and bridges/cages DNA together to form a nucleoprotein network in vivo [16][17][18][19][20][21][22][23] . This nucleoprotein complex recruits SMC to disentangle and organize replicated DNA 3,11,13,24,25 . ParB-parS also interacts with an ATPase ParA to power the segregation of replicated chromosomes [26][27][28][29][30] . Engineered strains harboring a nucleation-competent but spreading-defective mutant of parB are either unviable 10 or have elevated number of anucleate cells 4,7,8,15,[31][32][33][34] . Despite the importance of spreading for proper chromosome segregation, the mechanism by which a few parS-bound ParB can recruit hundreds more ParB molecules to the vicinity of parS to assemble a high molecular-weight nucleoprotein complex is not fully understood.Since the first report in 1995 35 , ParB spreading has been observed in vivo by chromatin immunoprecipitation in multiple bacterial species 12,[15][16][17]19,36 . The nucleation of ParB on parS has also been demonstrated in vitro 4,10,16,17,20,[37][38][39] , however parS-dependent ParB spreading has resisted biochemical reconstitution [17][18][19]40,41 . Unsuccessful attempts to reconstitute spreading in vitro suggests that additional factors might be missing. Recently, works by Soh et al (2019) and Osorio-Valeriano et al (2019) on Bacillus subtilis and Myxococcus xanthus ParB, respectively, showed that ParB binds and hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP), and that CTP modulates the binding affinity of ParB to parS 42,43 . A co-crystal structure of Bacillus ParB with CDP and that of a Myxococcus ParB-li...

show abstract

Ploidy is an important determinant of fluoroquinolone persister survival

Murawski

Brynildsen

2021

Current Biology

View full text Add to dashboard Cite

Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria

Cited by 9 publications

References 20 publications

Live cell imaging reveals that RecA finds homologous DNA by reduced dimensionality search

Live cell imaging reveals that RecA finds homologous DNA by reduced dimensionality search

ParB spreading on DNA requires cytidine triphosphatein vitro

Ploidy is an important determinant of fluoroquinolone persister survival

Contact Info

Product

Resources

About