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Characterization of Mu prophage lacking the central strong gyrase binding site: localization of the block in
1Department of Microbiology, University of Colorado Health Sciences Center, Denver 80262, USA.
Abstract:
Bacteriophage Mu contains an unusually strong DNA gyrase binding site (SGS), located near the center of its genome, that is required for efficient Mu DNA replication (M. L. Pato, Proc. Natl. Acad. Sci. USA 91:7056-7060, 1994; M. L. Pato, M. M. Howe, and N. P. Higgins, Proc. Natl. Acad. Sci. USA 87:8716-8720, 1990). Replication of wild-type Mu initiates about 10 min after induction of a lysogen, while replication in the absence of the SGS is delayed about an hour. To determine which step in the replication pathway is blocked in the absence of the SGS, we inactivated the SGS by deletion and by insertion and studied the effects of these alterations on various stages of Mu DNA replication. Following induction in the absence of a functional SGS, early transcription and synthesis of the Mu-encoded replication proteins occurred normally. However, neither strand transfer nor cleavage at the Mu genome termini could be detected 40 min after induction. The data are most consistent with a requirement for the SGS in the efficient synapsis of the Mu prophage termini to form a separate chromosomal domain.
Insights
The strong DNA gyrase binding site (SGS) is crucial for efficient bacteriophage Mu DNA replication. Its absence delays replication by blocking early steps like strand transfer and terminus cleavage.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage Mu possesses a unique DNA gyrase binding site (SGS) essential for its DNA replication.
- Efficient replication of wild-type Mu initiates approximately 10 minutes post-induction, whereas SGS-deficient replication is delayed by an hour.
Purpose of the Study:
- To investigate the specific replication stage impaired by the absence of the SGS.
- To elucidate the role of the SGS in bacteriophage Mu DNA replication.
Main Methods:
- Inactivation of the SGS through deletion and insertion mutations.
- Analysis of Mu DNA replication stages following induction in SGS-altered mutants.
Main Results:
- Early transcription and synthesis of replication proteins occurred normally in the absence of a functional SGS.
- Strand transfer and cleavage at Mu genome termini were not detected 40 minutes post-induction in SGS-deficient mutants.
Conclusions:
- The SGS is required for efficient synapsis of Mu prophage termini.
- The SGS facilitates the formation of a distinct chromosomal domain necessary for replication initiation.