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Biochim Biophys Acta Gen Subj. 2017 Sep;1861(9):2165-2174. doi: 10.1016/j.bbagen.2017.06.013. Epub 2017 Jun 16.

Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication.

Author information

1
Department of Molecular Biology, Rowan University, Stratford, NJ 08084, United States; Program in Biotechnology, Department of Medical Laboratory Sciences, College of Health Sciences, University of Delaware, Newark, DE 19716, United States.
2
Department of Molecular Biology, Rowan University, Stratford, NJ 08084, United States.
3
Department of Molecular Biology, Rowan University, Stratford, NJ 08084, United States. Electronic address: biswassb@rowan.edu.

Abstract

DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~15Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication.

KEYWORDS:

DNA binding; DnaA protein; FRET; Fluorescence Resonance Energy Transfer; Initiation of DNA replication

PMID:
28630006
DOI:
10.1016/j.bbagen.2017.06.013
[Indexed for MEDLINE]

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