Once sufficient single stranded DNA ssDNA has

Once sufficient single-stranded DNA (ssDNA) has been exposed at origins, synthesis of leading and lagging strands is initiated by the DNA polymerase α-primase complex (Pol α). Lagging-strand synthesis requires repeated cycles of Pol α-dependent priming and subsequent primer extension by Pol δ. Pol α first synthesizes 7–12 nucleotide (nt) RNA primers before transferring them to the DNA polymerase domain, where further extension to about 20–25 nt takes place (Pellegrini, 2012). Evidence suggests that Pol α must be functionally recruited to replication forks for efficient lagging-strand primer synthesis: priming on ssDNA by both human (Collins and Kelly, 1991) and yeast Pol α (Taylor and Yeeles, 2018) is inhibited by RPA; repeated lagging-strand priming by yeast Pol α is dependent on template unwinding by CMG (Georgescu et al., 2015). The details of this functional recruitment are yet to be elucidated. The mechanism by which continuous leading-strand replication is primed by Pol α at replication origins is currently unknown. Furthermore, in vivo studies in quick way yeast have reached conflicting conclusions regarding the location of leading-strand start sites relative to an origin. For example, one study concluded that the ARS1 origin contains a single leading-strand start site (Bielinsky and Gerbi, 1999). The site was located between the ARS consensus sequence (ACS), which forms part of a high-affinity ORC binding site required for MCM loading (Bell and Labib, 2016, Coster and Diffley, 2017), and the B2 element, a sequence element located downstream of the ACS that enhances origin activity (Chang et al., 2011, Marahrens and Stillman, 1992). However, a second study found that Pol α DNA synthesis peaked just upstream of the ACS, indicating that leading strands might be started outside the origin sequence, potentially from “lagging-strand” primers (Garbacz et al., 2018). Consequently, the relationship between origin sequences and leading-strand start sites is yet to be fully resolved. Pol ε is responsible for the bulk of leading-strand synthesis in vivo (Daigaku et al., 2015, Nick McElhinny et al., 2008, Pursell et al., 2007) and physically associates with CMG (Langston et al., 2014, Sengupta et al., 2013, Sun et al., 2015, Zhou et al., 2017). Furthermore, leading-strand synthesis rates matching those observed in vivo can only be attained by a reconstituted replisome when Pol ε is synthesizing the leading strand in conjunction with PCNA (Yeeles et al., 2017). Therefore, once the primer for leading-strand replication has been synthesized, the 3ʹ end must be coupled to CMG-bound Pol ε (CMGE) before rapid and efficient leading-strand replication can commence. Multiple non-mutually exclusive mechanisms might account for this process (Kunkel and Burgers, 2017). The simplest involves direct primer transfer from Pol α to CMGE. Support for this mechanism comes from observations that Pol α can prime the leading-strand template at model replication forks with CMG (Georgescu et al., 2015), and rapid and efficient leading-strand synthesis is observed in in vitro replication reactions where Pol α and Pol ε are the only DNA polymerases (Yeeles et al., 2017). In contrast, in vivo (Daigaku et al., 2015, Garbacz et al., 2018) and in vitro (Yeeles et al., 2017) experiments have indicated that, in addition to its role in lagging-strand synthesis, Pol δ might also participate in the initiation of leading-strand replication via a polymerase switch mechanism, with the 3ʹ end of the nascent leading strand sequentially transferred from Pol α to Pol δ to CMGE. Why such an elaborate mechanism may be required is unknown, as is the frequency by which the two pathways are utilized. In this study, we have addressed these outstanding questions by mapping start sites for leading-strand replication at two S. cerevisiae replication origins using a reconstituted replication system (Taylor and Yeeles, 2018, Yeeles et al., 2015, Yeeles et al., 2017), determining the basis of Pol α recruitment to these sites, and defining the pathway by which the 3ʹ end of the nascent leading strand is connected to CMGE following primer synthesis. This has enabled us to elucidate the mechanism of bidirectional leading-strand synthesis establishment at eukaryotic DNA replication origins.