br STAR Methods br Acknowledgments

STAR★Methods
Acknowledgments We thank J. Diffley for protein-expression strains and J. Sale, H. Williams, and members of the Yeeles lab for helpful discussions and comments on the manuscript. This work was supported by the Medical Research Council (MC_UP_1201/12).
Introduction A variety of means, including UV irradiation, dNTP depletion, and oncogene activation at precancerous lesions, induce replication stress, which causes replication errors if left undealt with (Hills and Diffley, 2014, Zeman and Cimprich, 2014). Eukaryotic DC260126 have developed the DNA replication checkpoint pathway to detect, signal, and repair DNA lesions caused by replication stress. Mutations in factors involved in DNA replication checkpoints lead to increased genome instability in both yeast and human cells (Ciccia and Elledge, 2010, Cimprich and Cortez, 2008, Huen and Chen, 2008, Maréchal and Zou, 2013, Yeeles et al., 2013). Moreover, replication errors likely play a prominent role in tumorigenesis in humans (Tomasetti and Vogelstein, 2015). Therefore, it is important to understand how the DNA replication checkpoint pathway deals with replicative stress.
Results To understand how Rad53 functions to maintain replisome integrity, we first analyzed newly synthesized leading- and lagging-strand DNA in wild-type (WT) and rad53-1 mutant cells using BrdU-IP-ssSeq, a method that detects synthesis of both leading and lagging strands (Yu et al., 2014). Briefly, yeast cells were synchronized in G1 phase using α factor and released into fresh medium containing the nucleotide analog BrdU and 0.2 M hydroxyurea (HU) (Figure 1A). HU depletes dNTP pools through inhibition of RNR and activates the DNA replication checkpoint. However, HU has no apparent effect on the initiation of DNA replication from early replication origins. Newly synthesized DNA marked by BrdU was immuno-precipitated using anti-BrdU antibodies and subjected to strand-specific sequencing (BrdU-IP-ssSeq). Sequence reads were mapped to both Watson and Crick strands of the yeast reference genome (Figure 1B). Inspection of BrdU-IP-ssSeq peaks at early replication origins in WT cells revealed that these peaks were largely symmetrical around replication origins (Figure 1B). These results suggest that DNA synthesis progressed bi-directionally from the origin for similar distances on both the leading and lagging strand, consistent with the idea that synthesis of leading and lagging strand is coupled. To analyze BrdU-IP-ssSeq quantitatively, we used a sliding window of 200 bp and calculated the average log2 ratio of sequence reads of Watson over Crick strands surrounding 134 early replication origins fired in the presence of HU. We observed that BrdU-IP-ssSeq peaks exhibited a small but consistent bias toward the leading strand (Figure 1C and Figure S1). The leading-strand bias indicates that lagging-strand synthesis is slower relative to leading-strand synthesis in WT cells. Based on the analysis of seven independent BrdU-IP-ssSeq datasets in WT cells (Figures S1A and S1B), nascent lagging-strand DNA length was 359 nt shorter than nascent leading strand, with the average 5.32 Kb BrdU peak length of 134 origins. This may be slightly underestimated, because there are slightly more Ts where BrdU is incorporated at the lagging strand (226,146 Ts) than the leading strand (220,433 Ts) in the replicated regions. This result suggests that a short stretch of single-stranded lagging template is likely exposed in WT cells (see Figure 2). Together, these results are consistent with the idea that synthesis of leading and lagging strands is coupled in WT cells, with a short ssDNA gap on the lagging-strand template (Figure 1D).