Modelling DNA replication fork stability and collapse using chromatin fiber analysis and the R-ODD-BLOBS program

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• DNA replication is a fundamental process, but its regulation and the role of associated proteins, especially during checkpoint activation or loss, are complex.
• Understanding replication fork dynamics is crucial for comprehending genome stability and disease.
• Schizosaccharomyces pombe is a model organism for studying cell cycle control and DNA replication.

Purpose of the Study:

• To characterize the anatomy of DNA replication forks in wild-type and mutant Schizosaccharomyces pombe strains.
• To investigate the distribution of Rad51 and Cdc45 proteins at replication forks and their correlation with DNA synthesis.
• To assess the impact of checkpoint loss (mrc1Δ and cds1Δ mutations) on replication fork structure and protein association.

Main Methods:

• Utilized BrdU labeling to measure synthesized DNA lengths at replication forks.
• Employed chromatin fiber imaging and pixel intensity analysis.
• Developed and applied the R-ODD-BLOBS computational tool for analyzing large datasets of chromatin spread data.
• Quantified Rad51 and Cdc45 protein colocalization with replicated and unreplicated DNA regions.

Main Results:

• Average BrdU tract lengths were measured: cds1Δ (~2.9 kb) > wild type (~2.5 kb) > mrc1Δ (~1.7 kb).
• Rad51 protein was found in 22% more replicated areas in mrc1Δ mutants compared to wild type.
• Cdc45 protein distribution was also analyzed in relation to replication fork status.

Conclusions:

• The R-ODD-BLOBS tool is effective for analyzing chromatin spread data to understand DNA replication.
• Checkpoint loss, particularly in mrc1Δ, alters replication fork structure and protein composition.
• Increased Rad51 presence in mrc1Δ mutants suggests a higher reliance on homologous recombination repair pathways.