br Results br Discussion In this study we have

Results
Discussion In this study, we have determined the pathway by which the 3ʹ end of the nascent leading strand is connected with CMGE after priming, revealing that Pol δ likely plays a crucial role in establishing all continuously synthesized leading strands at eukaryotic replication origins (Figures 1 and 2). Initiation-site mapping experiments have identified start sites for leading-strand replication at two S. cerevisiae origins. Synthesis is predominantly initiated outside the origin sequence; Left leading strands are started to the right, and Right leading strands are started to the left (Figures 3 and 4). This distribution strongly suggests that leading strands are established from lagging-strand primers synthesized at replication forks on opposite sides of the origin. We provide direct evidence to support this conclusion: first, delaying Pol α addition to reactions lengthened, rather than shortened, leading-strand products (Figure 5); second, the two replisomes remain interdependent downstream of CMG activation, because placing a single CPD in the lagging-strand template of the leftward fork blocked the establishment of rightward leading strands (Figure 6). The mechanism of priming at origins that we have uncovered provides a clear mechanistic basis for Pol δ function in establishing continuous leading-strand synthesis.
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 cells 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. In CH5138303 yeast, the DNA replication checkpoint kinase Rad53, which is equivalent to Chk1 in human cells, is activated via the upstream kinase Mec1 (ATR in human cells) to perform multiple functions, including the following three functions in response to DNA replication stress (Yeeles et al., 2013). First, Rad53 inhibits firing of late replication origins through phosphorylation of Sld3 and Dbf4 (Lopez-Mosqueda et al., 2010, Santocanale and Diffley, 1998, Zegerman and Diffley, 2010), proteins critical for the initiation of DNA replication. Second, Rad53 upregulates the levels of dNTPs through phosphorylation of the Dun1 kinase, which controls the degradation of Sml1, an inhibitor of ribonucleotide reductase (RNR) involved in the rate-limiting step of dNTP synthesis (Zhao and Rothstein, 2002). Activated Dun1 kinase also represses RNR gene transcription. Third, Rad53 prevents collapse of DNA replication forks under replication stress (Lopes et al., 2001, Sogo et al., 2002, Tercero and Diffley, 2001). Genetic evidence indicates that the essential function of Rad53 and Mec1 is linked to their role at DNA replication forks (Desany et al., 1998). Moreover, the essential function of Rad53 and Mec1 (as well as its counterpart, ATR) can be suppressed by elevated levels of dNTPs, including deletion of SML1 in yeast cells (Lopez-Contreras et al., 2015, Zhao et al., 1998). All these functions of DNA replication checkpoint kinases in the regulation of firing of late-replication origins, upregulation of dNTP synthesis, and maintenance of replisome functions are conserved in human cells. However, it remains largely unknown how DNA replication checkpoint kinases prevent fork collapse and how elevated dNTP levels help these kinases to perform their essential function. Early studies indicate that replisome components are reduced in checkpoint mutant cells under replication stress (Cobb et al., 2003), which leads to the model that DNA replication checkpoint kinases are required for the stable association of proteins with DNA replication forks. In contrast, recent studies indicate that replisome components remain associated with DNA replication forks in checkpoint mutant yeast cells (De Piccoli et al., 2012) and upon inhibition of checkpoint kinases in human cells (Dungrawala et al., 2015). Despite this, it is known that long stretches of single-stranded (ss) DNA are generated in yeast and human cells deficient in DNA replication checkpoint (Buisson et al., 2015, Sogo et al., 2002, Toledo et al., 2013). However, it is largely unknown how excessive ssDNA, which is detrimental to cells, is generated (Berens and Toczyski, 2012). Here, we employed strand-specific sequencing methods to analyze DNA synthesis at leading and lagging strands and the association of the ssDNA binding protein, replication factor A (RPA), with DNA replication forks in rad53-1 mutant cells. We observed that DNA synthesis proceeds much further along the lagging strand than the corresponding leading strand, resulting in the exposure of long stretches of ssDNA at the leading-strand template coated with RPA. Mechanistically, we show that replicative helicase MCM and leading-strand DNA polymerase Pol ε move further than the actual site of DNA synthesis, and elevation of dNTP levels suppresses the uncoupled DNA synthesis in rad53-1 mutant cells. Therefore, we propose that DNA replication checkpoint kinases function to couple leading- and lagging-strand DNA synthesis under replication stress, thereby preventing the generation of long stretches of ssDNA.