Crystallization and preliminary X-ray characterization of the eukaryotic replication terminator Reb1-Ter DNA complex

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• Reb1 protein in Schizosaccharomyces pombe regulates programmed replication and transcription termination.
• These processes occur at replication fork barriers (RFBs), involving Reb1 binding to termination (Ter) DNA sites.
• Reb1 coordinates the arrest of replication forks and transcription from opposite directions.

Purpose of the Study:

• To determine the structural basis of Reb1's function in replication and transcription termination.
• To provide insights into the mechanism of termination mediated by the Reb1 protein family.

Main Methods:

• The Reb1 DNA-binding and replication-termination domain was expressed in E. coli and purified.
• Crystallization of the Reb1 domain in complex with a 26-mer Ter DNA site was achieved using batch crystallization under oil.
• X-ray diffraction data were collected to 3.0 Å resolution from crystals grown in space group P2₁.

Main Results:

• High-quality crystals of the Reb1-DNA complex were obtained, requiring annealing to improve diffraction.
• Unit-cell parameters were determined: a = 68.9, b = 162.9, c = 71.1 Å, β = 94.7°.
• The study yielded the first structural information on the Reb1 protein family.

Conclusions:

• This structural study offers crucial insights into the mechanism of replication and transcription termination.
• Understanding Reb1's mechanism is vital for comprehending eukaryotic DNA processing and gene regulation.