Mcm2-7 is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of its DNA Gate

Genome wide characterization of Mcm2-7 localization and origin activity for mrc1 and mcm mutant alleles. Overall design: Samples used in this study: alpha-factor arrested samples Mcm2p ChIP: wild type (UPY493) 1x mcm2DENQ (UPY524) 1x Mcm2-7 ChIP: wild type (UPY493) 1x mcm2DENQ (UPY524) 1x Hu Arrested samples BrdU Chip: wild type (UPY493) 2x mcm2DENQ (UPY524) 2x Mcm2-7 ChIP: wild type (UPY493) 2x mcm2DENQ (UPY524) 2x mrc1? (UPY722) 2x Rationale. To assay the effects of origin activation, cultures were arrested in G1, released into BrdU containing media, and subsequently processed for ChIP. To assay the efficacy of late firing origins, cultures were synchronized in G1 as above, released into Hu + BrdU containing media, and processed for ChIP Experimental Outline. Chromatin immunoprecipitation (ChIP) was performed as described (Aparicio et al., 1997; Katou et al., 2003) using yeast engineered for BrdU uptake (Vernis et al., 2003). Sequencing libraries were generated from the above immunoprecipitated DNA using the Illumina sample prep kit, multiplexed, and sequenced on a GAII Illumina sequencer. Genome Assembly. For each sample sequenced reads were aligned to the S. cerevisiae S288C reference genome (SacCer2, SGD June 2008) (Kent et al., 2002) using MAQ (Li et al., 2008) allowing up to three mismatched bases. Once mapped, only those reads with a Phred quality score of 35 or greater were used for downstream analysis. For each experiment, reads were binned across the genome, and RPKM (reads per kilobase per million mappable reads) was calculated for each bin. For all of the BrdU ChIP-Seq experiments, bins of 5000 bp stepping every 1250 bp were used, and for all the Mcm ChIP-Seq experiments, bins of 1000 bp stepping every 250 bp were used. All in silico analyses were done in the R programming environment using the Bioconductor suite of packages (Gentleman et al., 2004; RDevelopmentCoreTeam, 2010). Experimental replicates were quantile normalized and combined by calculating the mean score within each bin. Peak determination. For each bin along a chromosome, a probability was assigned according to that chromosome’s empirical cumulative distribution of RPKM. A threshold was determined, and bins with a probability greater than or equal to the cutoff were taken as peaks (the ranges of overlapping peaks were merged such that the resulting peaks represented the union of all enriched bins). For BrdU ChIP-Seq in HU arrested cells, the thresholds used were as follows: WT strains p = 0.95, Mrc1 p = 0.75, and Mcm2DENQ p =0.75. For ChIP-Seq experiments in G1 arrested cells, a threshold of p = 0.96 was used. Replicates were combined by taking the intersection of peak calls. When possible, peaks were assayed for concordance against analogous data previously reported (Crabbe et al., 2010).