A Nucleotide Switch as the Predominant Mechanism Preventing Re-initiation of DNA Replication in Escherichia coli


            The process of chromosomal DNA replication in all living organisms is a highly regulated process, ensuring that a round of DNA replication occurs once, and only once, per associated cell cycle.  In the gram-negative bacterium, Escherichia coli, initiation of DNA replication is triggered at a single DNA origin, oriC, by the initiator protein, DnaA.  Three cellular mechanisms are thought to participate in a complementary manner to maintain tight control over this process.  These include: the physical sequestration of oriC by SeqA protein, the titration of DnaA away from the origin by the datA chromosomal locus, and the accelerated hydrolysis of active ATP-DnaA, to inactive ADP-DnaA, termed RIDA (regulatory inactivation of DnaA).  The recent discovery of the novel Hda protein as a necessary component of RIDA enabled the further investigation of this activity in vivo.  Although Hda-deficient (Dhda) E. coli cells continue to grow and survive, flow cytometric analysis revealed that a lack of functional Hda results in asynchronous initiation events and over-initiation, indicating a loss of tight control over the initiation process.  In addition, the extent of over-initiation in E. coli cells engineered to be deficient in one of the known DNA replication control mechanisms (Dhda, DseqA, DdatA) was investigated by quantitative genomic DNA microarray analysis.  The results indicate that the DnaA nucleotide switch mechanism, which requires Hda, is the predominant mechanism preventing inappropriate re-initiation events at oriC.  Further flow cytometric and light scattering analyses of SeqA-deficient (DseqA) E. coli cells indicate an increased cell size in conjunction with increased DNA content, supporting a model in which SeqA plays a more dominant role at the levels of chromosomal DNA organization and segregation in the cell.


Name: Johanna Camara

Mentor: Michael J. Welch

Division: Analytical Chemistry

Laboratory: Organic Analytical Methods Group

Room/Building: A153/227

Mail stop: 8392

Telephone #: 301-975-4672

Fax #: 301-977-0685

E-mail: johanna.camara@nist.gov

Sigma Xi member: No

Category: Biology