S. pombereplication protein Cdc18 (Cdc6) interacts with Swi6 (HP1) heterochromatin protein

Abstract

Heterochromatin in S. pombe is associated with gene silencing at telomeres, the mating locus and centromeres. The compact heterochromatin structure raises the question how it unpacks and reforms during DNA replication. We show that the essential DNA replication factor Cdc18 (CDC6) associates with heterochromatin protein 1 (Swi6) in vivo and in vitro. Biochemical mapping and mutational analysis of the association domains show that the N-terminus of Cdc18 interacts with the chromoshadow domain of Swi6. Mutations in Swi6 that disrupt this interaction disrupt silencing and delay replication in the centromere. A mutation cdc18-I43A that reduces Cdc18 association with Swi6 has no silencing defect at the centromere, but changes Swi6 distribution and accelerates the timing of centromere replication. We suggest that fine tuning of Swi6 association at replication origins is important for negative as well as positive control of replication initiation.

Condensin Promotes Position Effects within Tandem DNA Repeats via the RITS Complex

“…We propose that early replication of strongly silenced peri-centromeric repeats allows the establishment of the position effect at an early stage so as to create a chromatin environment that can ensure the proper positioning of CENP-A. Consistent with this idea, the core region of centromeres, cnt , and the flanking imr regions, both of which are weakly silenced, replicate later than the highly heterochromatic otr regions (Li et al, 2011b). …”

“…BrdU IP was essentially performed as described previously (Li et al, 2011b). Primers used in this study are listed in Table S2.…”

Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase

“…In this context of generally repressive chromatin, specific mechanisms have recently been identified in yeasts that act to stimulate the early replication of specific sequences. For example, centromeres are early replicating in both budding and fission yeasts, though remarkably, this is achieved through distinct mechanisms that act to efficiently recruit the replication initiation kinase DDK to specific origins (Hayashi et al 2009;Li et al 2011;Natsume et al 2013). In Saccharomyces cerevisiae, the Forkhead Box (FOX) DNA binding proteins Fkh1 and Fkh2 are responsible for stimulating the activation of most noncentromeric, early-firing origins in the genome (Knott et al 2012).…”

Linking DNA replication to heterochromatin silencing and epigenetic inheritance

“…Biochemical mapping and mutational analysis demonstrate that the N-terminus of Cdc18 interacts with the chromoshadow domain of Swi6. Interestingly, mutations in the binding domain of Cdc18 and Swi6 alter the distribution of Swi6 at specific centromere regions as well as the timing of centromere replication [101]. Using a yeast twohybrid assay in combination with chromatin immunoprecipitation, HP1 has been found to interact with Cdc6, Cdc45, and RPA in mouse cells [102].…”

“…For instance, Mcm10 physically interacts with Sir2 in budding yeast [97 -99], and Drosophila Mcm10 interacts with HP1 [100]. In S. pombe, Cdc18 (ScCdc6) interacts with Swi6 (HP1) [101]. Biochemical mapping and mutational analysis demonstrate that the N-terminus of Cdc18 interacts with the chromoshadow domain of Swi6.…”