Rif1 Regulates Origin Firing and Telomere Length Through Distinct Mechanisms

In Saccharomyces cerevisiae, RIF1 negatively regulates telomere length, but the mechanism of this regulation remains unclear. We sought to elucidate Rif1 telomere length regulation. We first assessed whether the role of Rif1 in origin firing determined how it regulates telomere length. Rif1 recruits PP1 to block origin firing, and we generated a mutant that disrupted PP1 recruitment, rif1-pp1bs. We found rif1-pp1bs increased telomeric origin firing like rif1∆, but rif1-pp1bs telomeres were wild type. We next over-expressed a cell-cycle stabilized DDK, which antagonizes PP1 to promote origin firing. This construct, ∆N-dbf4 Cdc7 o/e, increased origin firing, but telomeres remained wild type. We expressed a third mutant, sld3-A bob1, which bypasses regulation of the pre-replication complex. The sld3-A bob1 mutant drastically increased firing at most origins; however, telomeres remained wild type. Finally, we tested RIF1 mutants with long telomeres, and found these mutants properly regulated origin firing. These data demonstrate that the role of Rif1 in origin firing is separable from its role in telomere length regulation. We next asked which regions of Rif1 are sufficient for its telomere function. We fused regions of Rif1 to the Gal4 DNA binding domain, and recruited this protein to Gal4 binding sites engineered next to a single telomere. We then assessed telomere length of the single telomere compared to the bulk telomeres. We identified a region of Rif1, from aa 177-996, which, when tethered to the telomere, can regulate telomere length at the single telomere but not at bulk telomeres. This region had been proposed to bind DNA through several critical lysines, but the positive charge of these lysines was essential even when this region was artificially tethered to the telomere. We then identified and mutated additional conserved residues in Rif1177-996. We narrowed down the functional region to aa 437 to 577, and showed the importance of several charged and uncharged residues in telomere length regulation. This region is comprised of HEAT repeats, a structure known to be important for protein interaction. We propose that Rif1 binds a partner protein through the HEAT repeats of aa 437 to 577 to promote telomere regulation