Identification of temperature-sensitive dnaD mutants of Staphylococcus aureus that are defective in chromosomal DNA replication

The DnaD protein in Gram-positive bacteria is thought to be essential for the initiation step in DNA replication. In the present study, we characterized two Staphylococcus aureus mutants whose temperature-sensitive growth phenotype could be complemented by a plasmid carrying the dnaD gene. These mut...

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Bibliographic Details
Published in:Molecular genetics and genomics : MGG 2004-05, Vol.271 (4), p.447-457
Main Authors: Li, Y, Kurokawa, K, Matsuo, M, Fukuhara, N, Murakami, K, Sekimizu, K
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Bacterial Proteins - genetics
Blotting, Southern
Chromosomes, Bacterial - genetics
Deoxyribonucleic acid
DNA
DNA Damage
DNA Repair
DNA Replication
DNA, Bacterial - genetics
DNA, Bacterial - metabolism
DNA-Binding Proteins - deficiency
DNA-Binding Proteins - genetics
Flow Cytometry
Genetics
Mitomycin - pharmacology
Mutation
Nucleic Acid Synthesis Inhibitors - pharmacology
Phenotype
Proteins
Staphylococcus aureus
Staphylococcus aureus - genetics
Staphylococcus aureus - growth & development
Temperature
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Summary:The DnaD protein in Gram-positive bacteria is thought to be essential for the initiation step in DNA replication. In the present study, we characterized two Staphylococcus aureus mutants whose temperature-sensitive growth phenotype could be complemented by a plasmid carrying the dnaD gene. These mutants each had a single amino acid substitution in the DnaD protein and showed decreased DNA synthesis at restrictive temperature. Analyses of the origin to terminus ratio by Southern blotting, and of origin numbers per cell by flow cytometry, revealed that, at the restrictive temperature, one mutant continued ongoing DNA replication but failed to initiate DNA replication. The other mutant, in contrast, could not complete ongoing DNA replication and proceeded to degrade the chromosome. However, if protein synthesis was inhibited, the second mutant could complete DNA replication. These results suggest that DnaD protein is necessary not only for the initiation step, but also to avoid replication fork blockage. Moreover, both mutants were sensitive to mitomycin C, a drug that induces DNA damage, suggesting that the DnaD protein is also involved in DNA repair.
ISSN:1617-4615
1617-4623
DOI:10.1007/s00438-004-0996-6