Cohesion establishment by yeast Replication Factor C (RFC) complexes

Abstract

Concomitant with DNA replication, sister chromatid cohesion is established to ensure faithful chromosome segregation. This is mediated by the ring-shaped cohesin complex. In Saccharomyces cerevisiae, cohesin is loaded onto chromosomes during G1 but its residence on DNA is not long-lasting. During S phase, the cohesin subunit Smc3 is acetylated by the acetyltrans¬ferase Eco1 to achieve long-lasting residence on DNA. This reaction is facilitated by multiple replication fork factors including the alternative PCNA loader RFCCtf18 complex, a member of the PCNA-RFC family of clamp loaders/unloaders that also include PCNA loader RFCRfc1 and PCNA unloader RFCElg1. RFCCtf18 is an enigmatic component of the replication machinery that is known to interact with leading strand Pol ε and has important roles in both establishing sister chromatid cohesion and maintaining replication checkpoint. How RFCCtf18 facilitates cohesion establishment, and whether PCNA loading is involved, remain unresolved. I reveal that PCNA loading by RFCCtf18 is required for cohesion establishment separately from its checkpoint function and independent from its interaction with Pol ε. I uncover post-replication lagging strand PCNA is important for cohesion establishment. I also determined that the prototypic PCNA loader RFCRfc1 is not a cohesion establishment factor, but is instead required for replication progression. Through a strand-specific approach, I reveal that RFCCtf18 and RFCRfc1 preferentially bind to and load separate pools of PCNA on the leading and lagging strands respectively. Rfc1 and Ctf18 therefore play divergent strand-specific roles at the replication fork. Loaded pools of PCNA on the leading and lagging strands are unequal and confer distinct molecular functions. Since lagging strand RFCRfc1 is not important for cohesion, and that leading strand RFCCtf18 is, I propose more complex mechanisms for RFCCtf18’s role in cohesion establishment.