Diversification of DNA-binding specificity via permissive and specificity-switching mutations in the ParB/Noc protein family

Jalal, Adam S. B.; Tran, Ngat T.; Stevenson, Clare E. M.; Chan, Elliot W.; Lo, Rebecca; Tan, Xiao; Noy, Agnes; Lawson, David M.; Le, Tung B. K.

doi:10.1101/724823

Cited by 7 publications

(7 citation statements)

References 74 publications

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“…Interestingly, our analysis of available ChIP-seq data (including the 17 chromosomal ParBs from various species produced in E. coli [142] and tested for DNA binding in this heterologous host, as well as two ParBs from V. cholerae and Corynebacterium glutamicum tested in their native hosts) showed that binding to parS half-sites GTTCCAC and GTTTCAC is not a unique feature of P. aeruginosa ParB (Figure 3a,b). ParB of P. aeruginosa and five other ParBs clearly bind to these heptanucleotides even in a heterologous host.…”

Section: Parb Binding To Half-pars: a Novel Aspect Of Parb-dna Interamentioning

confidence: 96%

Rules and Exceptions: The Role of Chromosomal ParB in DNA Segregation and Other Cellular Processes

et al. 2020

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The segregation of newly replicated chromosomes in bacterial cells is a highly coordinated spatiotemporal process. In the majority of bacterial species, a tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target(s) parS sequence(s), facilitates the initial steps of chromosome partitioning. ParB nucleates around parS(s) located in the vicinity of newly replicated oriCs to form large nucleoprotein complexes, which are subsequently relocated by ParA to distal cellular compartments. In this review, we describe the role of ParB in various processes within bacterial cells, pointing out interspecies differences. We outline recent progress in understanding the ParB nucleoprotein complex formation and its role in DNA segregation, including ori positioning and anchoring, DNA condensation, and loading of the structural maintenance of chromosome (SMC) proteins. The auxiliary roles of ParBs in the control of chromosome replication initiation and cell division, as well as the regulation of gene expression, are discussed. Moreover, we catalog ParB interacting proteins. Overall, this work highlights how different bacterial species adapt the DNA partitioning ParAB-parS system to meet their specific requirements.

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Section: Parb Binding To Half-pars: a Novel Aspect Of Parb-dna Interamentioning

confidence: 96%

Rules and Exceptions: The Role of Chromosomal ParB in DNA Segregation and Other Cellular Processes

et al. 2020

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“…N-terminally His-tagged MBP-tagged ParB (orthologous proteins from various bacterial species) were expressed from E. coli Rosetta (DE3) harboring pET-His-MBP-TEV-DEST::ParB plasmids (Table S1). His6-MBP-ParB proteins were purified via a one-step Ni-affinity column as described previously 56 .…”

Section: Protein Overexpression and Purificationmentioning

confidence: 99%

ParB spreading on DNA requires cytidine triphosphatein vitro

Jalal

Tran

2019

Preprint

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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...

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“…Here, the ability to assess many mutations in parallel and measure even subtle differences in affinity suggests that effects of mutations in trans could be compensated for by concomitant changes in nucleotides flanking cis-regulatory elements to preserve transcriptional responses (Beltran, et al, 2004;Rogers and Bulyk, 2018). In addition, the observation that mutations may stabilize helical conformations to promote DNA binding suggests that these residues could allow formation of permissive binding intermediates (Bloom, et al, 2010;Gong, et al, 2013;Jalal, et al, 2019;McKeown, et al, 2014;Starr, et al, 2017). The higher affinity binding of these permissive binding intermediates could allow TFs to acquire and accommodate additional TF or DNA mutations that would otherwise reduce binding to non-physiological levels, making otherwise inaccessible evolutionary trajectories feasible.…”

Section: Discussionmentioning

confidence: 99%

High-throughput binding affinity measurements for mutations spanning a transcription factor-DNA interface reveal affinity and specificity determinants

Aditham

Markin

Mokhtari

et al. 2020

Preprint

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Transcription factors (TFs) bind regulatory DNA to control gene expression, and mutations to either TFs or DNA can alter binding affinities to rewire regulatory networks and drive phenotypic variation. While studies have profiled energetic effects of DNA mutations extensively, we lack similar information for TF variants. Here, we present STAMMP (Simultaneous Transcription Factor Affinity Measurements via Microfluidic Protein Arrays), a highthroughput microfluidic platform enabling quantitative characterization of hundreds of TF variants simultaneously. Measured affinities for ~210 mutants of a model yeast TF (Pho4) interacting with 9 oligonucleotides (>1,800 Kds) reveal that many combinations of mutations to poorly conserved TF residues and nucleotides flanking the core binding site alter but preserve physiological binding, providing a mechanism for mutations in cis and trans to rewire networks without insurmountable evolutionary penalties. Moreover, biochemical double-mutant cycles across the TF-DNA interface reveal molecular mechanisms driving recognition, linking sequence to function.

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Diversification of DNA-binding specificity via permissive and specificity-switching mutations in the ParB/Noc protein family

Cited by 7 publications

References 74 publications

Rules and Exceptions: The Role of Chromosomal ParB in DNA Segregation and Other Cellular Processes

Rules and Exceptions: The Role of Chromosomal ParB in DNA Segregation and Other Cellular Processes

ParB spreading on DNA requires cytidine triphosphatein vitro

High-throughput binding affinity measurements for mutations spanning a transcription factor-DNA interface reveal affinity and specificity determinants

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