Yeast replication in the presence of MMS: WT and isw2 nhp10 strains

The eukaryotic DNA replication machinery must traverse every nucleosome present in the eukaryotic genome during S phase. Since nucleosomes are generally inhibitory to DNA-dependent processes, it is thought that chromatin structure must undergo extensive reorganization to facilitate DNA synthesis. However, the identity of chromatin-remodeling factors involved in replication and how they affect DNA synthesis is largely unknown. Here we show that two ATP-dependent chromatin-remodeling complexes in Saccharomyces cerevisiae, Isw2 and Ino80, function in parallel to promote DNA replication, especially during periods of replication stress. The rate of replication-fork progression is slowed when both chromatin-remodeling pathways are compromised. Both Isw2 and Ino80 complexes are recruited to actively replicating chromatin suggesting that these chromatin-remodeling complexes act directly to promote replication-fork progression. These findings support an important role for ATP-dependent chromatin-remodeling complexes in promoting DNA replication, and define specific stages of replication that require remodeling activity for normal function. A critical part of this study was the use of microarrays to measure DNA replication progression throughout the genome during MMS treatment. These samples are from experiments where we competed newly replicated DNA against unreplicated DNA on the same microarrays. Keywords: Time course Genomic DNA isolated from S.cerevisiae samples taken at 30, 45, 60, 90, and 120 minutes (wild type) or 45, 60, 90, 120, and 150 minutes (isw2 nhp10) after release from G1 arrest (in media containing 0.015% MMS) was digested by restriction enzyme and fractionated into 2 pools: DNA that was replicated in the latest S-phase, and DNA that had not yet been replicated. DNA from each pool was oppositely labeled and co-hybridized to a microarray. 19 total samples: 10 for WT collection 9 for isw2 nhp10 collection All samples were from the same 2 experiments (1 WT collection, 1 isw2 nhp10 collection) Each sample has one dye swap pair (with the exception of the 45 min sample from the isw2 nhp10 collection). Dye swapped samples are named identically until the final character (either a or b) Percent replication values were determined for each corresponding spot on the array as described within.