Clamp loading on the leading strand during DNA replication and initial reconstitution of sister chromatid cohesion establishment
Accurate and efficient replication of genetic material is essential to all life, with dysregulation of this vital process resulting in genomic instability and disease. In eukaryotes this process is performed by the replisome, a complex protein machinery composed of a core helicase, DNA polymerases, and a wide range of replisome-associated factors. One such factor is the sliding clamp PCNA, which acts as both a processivity factor to support fast and efficient DNA synthesis and as a recruitment platform in various genomic stability maintenance processes downstream of DNA replication. Regulation of chromatin bound PCNA is therefore also essential, making it important to understand the clamp loader complexes responsible for this regulation. The clamp loader Rfc1-RFC is essential for PCNA loading to support DNA replication, with an alternative clamp loader, Ctf18-RFC, instead important in many genomic stability maintenance processes. However, our understanding of PCNA loading during DNA replication is not complete, and the pleiotropic roles of the alternative clamp loader Ctf18-RFC are even less well understood. The work presented here uses *in vitro* reconstituted DNA replication systems to demonstrate a novel mechanism of PCNA loading by Ctf18-RFC. This supports a model for PCNA loading during DNA replication in which both clamp loader complexes are required, as well as providing insight into the requirement for Ctf18-RFC in various genomic stability processes. Once replicated, it is also essential that genetic material is faithfully divided between daughter cells. This requires sister chromatid cohesion, wherein the ring-shaped protein cohesin binds sister chromatids in a pairwise manner until cell division. Cohesion establishment is thought to be coupled to DNA replication and involves various replisome associated factors, however mechanistic understanding is lacking, limited by the indirect nature of cytological approaches. The work presented here has therefore made steps to reconstitute DNA replication-coupled cohesion establishment *in vitro*, a tool that would prove invaluable for understanding this complex process.
Clamp loader, Cohesin, Cohesion, Ctf18-RFC, DNA replication, PCNA
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