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Diversification of DNA-Binding Specificity by Permissive and Specificity-Switching Mutations in the ParB/Noc Protein Family.

ABSTRACT: Specific interactions between proteins and DNA are essential to many biological processes. Yet, it remains unclear how the diversification in DNA-binding specificity was brought about, and the mutational paths that led to changes in specificity are unknown. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB [Partitioning Protein B] and Noc [Nucleoid Occlusion Factor], which both play roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity and that mutational paths to new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc and, in a broader context, might be useful for understanding the evolution of other classes of DNA-binding proteins.

SUBMITTER: Jalal ASB

PROVIDER: S-EPMC7383237 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description:Specific interactions between proteins and DNA are essential to many biological processes. Yet it remains unclear how the diversification in DNA-binding specificity was brought about, and what were the mutational paths that led to changes in specificity. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB and Noc: both having roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity, and that mutational paths to a new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc, and in a broader context, might be useful in understanding the evolution of other classes of DNA-binding proteins.

2019-08-05 | GSE129285 | GEO

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

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

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Permissive zones for the centromere-binding protein ParB on the Caulobacter crescentus chromosome.

Project description:Proper chromosome segregation is essential in all living organisms. In Caulobacter crescentus, the ParA-ParB-parS system is required for proper chromosome segregation and cell viability. The bacterial centromere-like parS DNA locus is the first to be segregated following chromosome replication. parS is bound by ParB protein, which in turn interacts with ParA to partition the ParB-parS nucleoprotein complex to each daughter cell. Here, we investigated the genome-wide distribution of ParB on the Caulobacter chromosome using a combination of in vivo chromatin immunoprecipitation (ChIP-seq) and in vitro DNA affinity purification with deep sequencing (IDAP-seq). We confirmed two previously identified parS sites and discovered at least three more sites that cluster ?8 kb from the origin of replication. We showed that Caulobacter ParB nucleates at parS sites and associates non-specifically with ?10 kb flanking DNA to form a high-order nucleoprotein complex on the left chromosomal arm. Lastly, using transposon mutagenesis coupled with deep sequencing (Tn-seq), we identified a ?500 kb region surrounding the native parS cluster that is tolerable to the insertion of a second parS cluster without severely affecting cell viability. Our results demonstrate that the genomic distribution of parS sites is highly restricted and is crucial for chromosome segregation in Caulobacter.

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DNA binding specificity for the bHLH-PAS transcription factor family

Project description:Transcription factor-DNA interactions and their specificities have been described for many different classes of transcription factor families. However, heterodimeric transcription factor complexes still remain poorly characterised. The basic-Helix-Loop-Helix (bHLH) transcription factor family is one of the largest transcription factor families that typically bind DNA though a degenerate CANNTG elements as heterodimers or homodimers. Here we characterise the DNA binding of the bHLH - Per-Arnt-Sim (PAS) (bHLH-PAS) domain containing transcription factor family using SELEX-high-throughput sequencing coupled with quantitative computational modelling analysis. We show that most dimeric bHLH-PAS transcription factors bind to distinct core NNCGTG response elements but bind over a much larger footprint than previously characterised. Modelled DNA-protein interactions were found to correlate with structural analysis, DNA shape predictions and in vivo transcription factor occupancy.

2022-07-02 | GSE159989 | GEO

Permissive zones for the centromere-binding protein ParB on the Caulobacter crescentus chromosome

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Structural insights into the DNA-binding specificity of E2F family transcription factors.

Project description:The mammalian cell cycle is controlled by the E2F family of transcription factors. Typical E2Fs bind to DNA as heterodimers with the related dimerization partner (DP) proteins, whereas the atypical E2Fs, E2F7 and E2F8 contain two DNA-binding domains (DBDs) and act as repressors. To understand the mechanism of repression, we have resolved the structure of E2F8 in complex with DNA at atomic resolution. We find that the first and second DBDs of E2F8 resemble the DBDs of typical E2F and DP proteins, respectively. Using molecular dynamics simulations, biochemical affinity measurements and chromatin immunoprecipitation, we further show that both atypical and typical E2Fs bind to similar DNA sequences in vitro and in vivo. Our results represent the first crystal structure of an E2F protein with two DBDs, and reveal the mechanism by which atypical E2Fs can repress canonical E2F target genes and exert their negative influence on cell cycle progression.

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Evolution of the B3 DNA binding superfamily: new insights into REM family gene diversification.

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Project description:Epigenetic modifications such as DNA methylation have large effects on gene expression and genome maintenance. Helicobacter pylori, a human gastric pathogen, has a large number of DNA methyltransferase genes, with different strains having unique repertoires. Previous genome comparisons suggested that these methyltransferases often change DNA sequence specificity through domain movement--the movement between and within genes of coding sequences of target recognition domains. Using single-molecule real-time sequencing technology, which detects N6-methyladenines and N4-methylcytosines with single-base resolution, we studied methylated DNA sites throughout the H. pylori genome for several closely related strains. Overall, the methylome was highly variable among closely related strains. Hypermethylated regions were found, for example, in rpoB gene for RNA polymerase. We identified DNA sequence motifs for methylation and then assigned each of them to a specific homology group of the target recognition domains in the specificity-determining genes for Type I and other restriction-modification systems. These results supported proposed mechanisms for sequence-specificity changes in DNA methyltransferases. Knocking out one of the Type I specificity genes led to transcriptome changes, which suggested its role in gene expression. These results are consistent with the concept of evolution driven by DNA methylation, in which changes in the methylome lead to changes in the transcriptome and potentially to changes in phenotype, providing targets for natural or artificial selection.

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