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Structure and function of transcription-repair coupling factor. I. Structural domains and binding properties
1Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
The Journal of Biological Chemistry
|March 3, 1995
Summary
The Mfd protein (transcription-repair coupling factor, TRCF) in E. coli links DNA transcription to repair. It interacts with RNA polymerase and UvrA to facilitate DNA damage repair during transcription.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Bacterial Genetics
Background:
- The mfd gene product (130-kDa) in Escherichia coli is crucial for coupling transcription with DNA repair.
- Mfd (transcription-repair coupling factor, TRCF) displaces stalled RNA polymerase (Pol) at DNA lesions.
- TRCF enhances template strand repair rates during transcription by interacting with UvrA.
Purpose of the Study:
- To investigate the molecular interactions of TRCF with DNA, RNA Pol, and UvrA.
- To elucidate the functional domains of TRCF involved in transcription-repair coupling.
Main Methods:
- Biochemical assays to study TRCF binding to double-stranded DNA, single-stranded DNA, and RNA.
- Analysis of ATP-dependent DNA binding and dissociation.
- Structure-function analysis using specific TRCF residue regions.
Main Results:
- TRCF binds double-stranded DNA non-specifically, with DNA wrapping suggested by protection patterns.
- ATP binding is required for DNA binding, and ATP hydrolysis promotes TRCF dissociation from DNA and stalled RNA Pol.
- TRCF recognizes stalled elongation complexes via direct interaction with RNA Pol, independent of transcription bubbles.
Conclusions:
- TRCF utilizes distinct domains for interacting with DNA, RNA Pol, and UvrA.
- Residues 379-571 bind stalled RNA polymerase, the helicase motifs (571-931) bind ATP and duplex polynucleotides, and residues 1-378 bind UvrA.
- ATP hydrolysis is essential for TRCF's dissociation and function in DNA repair.