Elevated recombination rates in transcriptionally active DNA

Area of Science:

• Molecular Biology
• Yeast Genetics
• DNA Recombination

Background:

• Recombination between repeated DNA sequences is a critical process in genome stability.
• Transcription, particularly RNA polymerase II-dependent, is known to influence DNA recombination.
• The GAL10 gene in Saccharomyces cerevisiae provides a model system to study transcription-recombination interactions.

Purpose of the Study:

• To investigate the impact of RNA polymerase II-dependent transcription on direct repeat recombination within the GAL10 gene.
• To determine if constitutive expression of GAL10 gene repeats affects plasmid loss versus conversion events.
• To identify the role of transcription activators (GAL4) and repressors (GAL80) in modulating this recombination.

Main Methods:

• Utilized isogenic yeast strains with null mutations in GAL4 or GAL80.
• Constructed strains with integrated GAL10 gene repeats for recombination analysis.
• Measured recombination rates (plasmid loss and conversion) under constitutive and non-constitutive expression conditions.
• Employed Northern blot analysis to assess the role of transcription initiation sites.

Main Results:

• Constitutive expression of the GAL10 gene construct led to a 15-fold increase in the rate of plasmid loss.
• Conversion events that resulted in retention of the integrated plasmid were not stimulated by repeat expression.
• Analysis of promoter mutations indicated that transcription initiation within the integrated plasmid sequences mediated the observed recombination stimulation.

Conclusions:

• RNA polymerase II-dependent transcription significantly enhances recombination leading to plasmid loss between direct repeats.
• The stimulatory effect on recombination is dependent on transcription initiation occurring within the repeated sequences.
• Differential effects on plasmid loss versus conversion suggest distinct mechanisms are involved in these recombination outcomes.