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

ABSTRACT We describe the anatomy of replication forks by comparing the lengths of synthesized BrdU-labelled DNA in wild type, mrc1Δ and cds1Δ Schizoasaccharomyces pombe . We correlated Rad51 and Cdc45 proteins relative to their positions on the fork, replicated tract, or unreplicated regions. We did this by using chromatin fiber images. These fibers track pixel intensity data, which is analyzed using our program: R-ODD-BLOBS. We compared the lengths of BrdU tracts and proteins, as well as the percentage of Rad51 and Cdc45 colocalization, and compared our results with literature findings. We measured average BrdU lengths consistent with current literature; cds1Δ was the longest at ∼2.9 kb (8.6 pixels, px), wild type was ∼ 2.5 kb (7.5 px), and mrc1Δ was the shortest at ∼1.7 kb (5.1 px). Intriguingly, Rad51 was found at 22% more replicated areas in mrc1Δ than in wild type. This suggests that homologous recombination repair may be more common at mrc1Δ forks. In this study, we summarize the usefulness of a computational modeling tool to assess large datasets of chromatin spread data. In turn, we find patterns of DNA replication length and protein components at replication forks, to describe the anatomy of a fork and how structures change with checkpoint loss. Abstract Figure GRAPHICAL ABSTRACT: R-ODD-BLOBS uses chromatin fiber data to rigorously model replication fork structures. DNA replication forks are multi-subunit structures that must pair and regulate DNA copying activity of the polymerases with unwinding activity of helicase. Chromatin fiber data retains proteins, and can be used to detect DNA synthesis (blue) and associated DNA replication fork proteins such as MCM4 helicase (MCM4) and replication protein A (RPA). In our work, we have used homologous recombination protein Rad51 and helicase factor Cdc45 to understand how DNA replication fork structures are destabilized during hydroxyurea treatment, and how they fail to recover because of Cdc45/helicase mis-localization.