Polymerase-Usage Sequencing Identifies Initiation Zones With Less Bias Throughout S phase in Mouse Embryonic Stem Cells
In mammalian cells, DNA replication can initiate from various locations even in different cycles of the same cell of origin. Various methods have been developed to map replication initiation zones (IZs) genome-wide, often finding IZs far less than expected considering the replication fork speed and genome size. In particular, IZs for later stages of S phase are under-represented, potentially due to reduced initiation events at this stage, but also likely to be due to variability in the population, requiring higher sensitivity of the methods. However, to what degree these factors contribute to the data, have not been investigated in detail. Mouse ES cells present similar density of replication initiation events throughout S phase, unlike non-ES cells. Here, we re-analyzed IZs with respect to replication timing within S phase, using data sets previously obtained for mouse ES cells. These data sets identified over 5-fold more early IZs compared to late IZs, suggesting that more than 80% of late IZs are missed. In addition, by newly establishing cell lines to carry out polymerase-usage sequencing (Pu-seq), we mapped IZs genome-wide in mouse ES cells. Pu-seq presented less bias towards early IZs, suggesting that better sensitivity for identifying IZs in late S phase. Overall design: We mapped ribonucleotide-incorporation by the mutated DNA polymerase e (Pole1-M630F) at single-nucleotide resolution in mouse embryonic stem cells and subsequent informatics analysis was performed to generate datasets listed here.