Nonspecific DNA binding by P1 ParA determines the distribution of plasmid partition and repressor activities
| Autor: | Jamie Baxter, Barbara E. Funnell, William G. Waples |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
DNA Bacterial Operator Regions Genetic Mutation Missense Repressor DNA Primase DNA and Chromosomes Biochemistry DNA-binding protein Plasmid maintenance 03 medical and health sciences chemistry.chemical_compound Viral Proteins Plasmid Escherichia coli Protein–DNA interaction Bacteriophage P1 Molecular Biology 030102 biochemistry & molecular biology Chemistry Circular bacterial chromosome Escherichia coli Proteins Cell Biology Bacterial nucleoid Chromosomes Bacterial Cell biology 030104 developmental biology Amino Acid Substitution DNA |
| Zdroj: | J Biol Chem |
| ISSN: | 1083-351X |
| Popis: | The faithful segregation, or “partition,” of many low-copy number bacterial plasmids is driven by plasmid-encoded ATPases that are represented by the P1 plasmid ParA protein. ParA binds to the bacterial nucleoid via an ATP-dependent nonspecific DNA (nsDNA)-binding activity, which is essential for partition. ParA also has a site-specific DNA-binding activity to the par operator (parOP), which requires either ATP or ADP, and which is essential for it to act as a transcriptional repressor but is dispensable for partition. Here we examine how DNA binding by ParA contributes to the relative distribution of its plasmid partition and repressor activities, using a ParA with an alanine substitution at Arg(351), a residue previously predicted to participate in site-specific DNA binding. In vivo, the parA(R351A) allele is compromised for partition, but its repressor activity is dramatically improved so that it behaves as a “super-repressor.” In vitro, ParA(R351A) binds and hydrolyzes ATP, and undergoes a specific conformational change required for nsDNA binding, but its nsDNA-binding activity is significantly damaged. This defect in turn significantly reduces the assembly and stability of partition complexes formed by the interaction of ParA with ParB, the centromere-binding protein, and DNA. In contrast, the R351A change shows only a mild defect in site-specific DNA binding. We conclude that the partition defect is due to altered nsDNA binding kinetics and affinity for the bacterial chromosome. Furthermore, the super-repressor phenotype is explained by an increased pool of non-nucleoid bound ParA that is competent to bind parOP and repress transcription. |
| Databáze: | OpenAIRE |
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