The Role of Rif1 in telomere length regulation is separable from its role in origin firing

Abstract

To examine the established link between DNA replication and telomere length, we tested whether firing of telomeric origins would cause telomere lengthening. We found that RIF1 mutants that block Protein Phosphatase 1 (PP1) binding activated telomeric origins but did not elongate telomeres. In a second approach, we found overexpression of ∆N-Dbf4 and Cdc7 increased DDK activity and activated telomeric origins, yet telomere length was unchanged. We tested a third mechanism to activate origins using the sld3-A mcm5-bob1 mutant that deregulates the pre-replication complex, and again saw no change in telomere length. Finally, we tested whether mutations in RIF1 that cause telomere elongation would affect origin firing. We found that neither rif1-∆1322 nor rif1HOOK affected firing of telomeric origins. We conclude that telomeric origin firing does not cause telomere elongation, and the role of Rif1 in regulating origin firing is separable from its role in regulating telomere length.

Rif1 regulates telomere length through conserved HEAT repeats

“…Cells were grown logarithmically in yeast peptone dextrose (YPD) to an optical density of close to 1.0 described in (12). Cells were washed and resuspended in sterile water and 0.1M Lithium acetate (LiAc, Sigma L6883-250G) before pelleting.…”

“…Of note, PvuII and XhoI were both used together to digest genomic DNA for all Southern blots to visualize the 1L telomere (PvuII) and bulk telomere (XhoI) restriction fragments. To better visualize the 1L telomere, 10ng of 2-log ladder (NEB N3200L) was loaded instead of 100ng, as done previously (12). Digested gDNA and ladder were loaded onto a 1% agarose gel and electrophoresed overnight at ~47V in 1XTTE.…”

“…Protein extraction using trichloroacetic acid (TCA, Sigma T0699) and western blotting methods are similar to those described in (12). 500μL of cells were collected from overnight saturated yeast cultures grown in YPD.…”

“…The Rif1-PP1 complex blocks origin firing by de-phosphorylating Mcm4 in the pre-replication complex (7–11). We recently showed that this conserved Rif1 function does not regulate telomere length in yeast (12). The Rif1 C-terminus binds to Dbf4 (7,8), and also localizes Rif1 to the telomere through the Rap1 binding motif (RBM) (13), but deletion of these domains does not lead to telomeres that are as long as rif1Δ (12,13).…”

“…We recently showed that this conserved Rif1 function does not regulate telomere length in yeast (12). The Rif1 C-terminus binds to Dbf4 (7,8), and also localizes Rif1 to the telomere through the Rap1 binding motif (RBM) (13), but deletion of these domains does not lead to telomeres that are as long as rif1Δ (12,13). Thus, while we know several binding partners of Rif1, the domains and mechanism by which Rif1 negatively regulates telomere length in yeast remain elusive.…”