Analyzing DNA Replication Fork Stability and Collapse Using Chromatin Fiber Analysis and the R-ODD-BLOBS Program.
We describe the anatomy of replication forks by detecting proteins associated with DNA replication (Cdc45 and RPA), DNA damage (H2A.X), and DNA repair (Rad51) relative to tracts of synthesized, 5'-bromodeoxyuridine (BrdU)-labeled DNA on chromatin fiber images. These fibers track pixel intensity and positional data, which are analyzed using our program: R-ODD-BLOBS (One Dimensional Data Boolean Logic Operations Binning System in the programming language R). We studied the effect of threshold and signal smoothing for BrdU and protein tracts following hydroxyurea in wild type fission yeast ( Schizoasaccharomyces pombe ), compared to DNA replication checkpoint mutants  mrc1Δ  and  cds1Δ . We show that R-ODD-BLOBS allows robust analysis of BrdU lengths and that Rad51, Cdc45, RPA, and H2A.X show distinctive, checkpoint-dependent locations around replicated tract ends. Rad51 was found at 22% more replicated areas in  mrc1∆  than in wild type, suggesting that homologous recombination repair may be more common at  mrc1Δ  forks. Helicase detachment in  cds1∆  post-HU was indicated by Cdc45 enrichment in unreplicated chromatin close to putative forks. Similarly,  cds1Δ  fibers show that Rad51, RPA, and H2A.X are distributed upstream of replicated areas more than in wild type. Excitingly, we find that H2A.X is distributed asymmetrically around replication forks, suggesting that the fork complex is a barrier for DNA damage signal extension into replicated areas. Together, R-ODD-BLOBS analysis shows a rigorous, iterative computational analysis tool to assess large chromatin spread datasets. R-ODD-BLOBS finds patterns of DNA replication length and protein components at replication forks that describe the anatomy of a fork and how structures change after replication checkpoint loss.
Cheng K, Aliar K, Manshaei R, Forsburg SL, Mazalek A, Sabatinos SA