MATERIALS AND METHODS

Yeast strains and genetics

Standard genetic methods for budding yeast strains construction and crossing were used (Shyian et al., 2016).

1D and 2D gels and Southern blot

2D gels were performed essentially as in (Shyian et al., 2016) using BglII enzyme for genomic DNA digestion. For 1D gels the first dimension gel was directly transferred to nylon membrane and probed with a radioactively labeled probe specific to Fob1-RFB site (Shyian et al., 2016).

rDNA instability assay (ADE2 marker loss)

ADE2 maker loss assays were performed essentially as in (Shyian et al., 2016).

Serial dilution spot assay

125 Serial dilution spot assays were performed essentially as in (Shyian et al., 2016).

Mcm4 and Cdc45 ChIP

Mcm4-Myc and Cdc45-Myc anti-Myc ChIP assays were performed essentially as in (Mattarocci et al., 2014).

‘Cowcatcher’ screens

Strains containing single copy of ADE2 and URA3 genes inserted into rDNA array were used for mutagenesis with EMS at 50% survival. EMS treated cultures were split in 10 separate tubes, inoculated into SC-ADE-URA and grown overnight (to counter select mutations in ADE2 and URA3). Then, aliquots were inoculated into YPAD and grown overnight (in order to let cell loose makers from rDNA). Dilutions were plated on 5-FOA plates (selection of URA3 loss) and incubated as in “ADE2 loss assay”. After visual inspection, red sectored colonies from 5-FOA plates were manually selected and their white sectors were streaked sequentially 2 times onto SC plates. Of ca. 50’000 colonies from 5-FOA plates, 30 independent reproducibly highly sectoring isolates were chosen. Those were back-crossed, sporulated, dissected and assessed for segregation of the high sectoring phenotype.

Isolates showing 2:2 segregation of sectoring (as Mendelian monoallelic mutations) were subjected to causative mutation identification using pooled linkage analysis, as in (Birkeland et al., 2010; Lang et al., 2015). Briefly, 20 spore colonies with sectoring phenotype were pooled together in one pool (+phenotype) and 20 white spore colonies were pooled in second pool (-phenotype); genomic DNA was isolated with Qiagen genomic tip kit. Total genomic DNA of the two pools was submitted to iGE3 Genomics Platform of University of Geneva for fragmentation, library preparation and whole genome deep sequencing. The FASTQ files with sequencing reads from iGE3 Genomics Platform were mapped to S.

cerevisiae genome with Mapping tool of the ‘HTSstation’ (http://htsstation.epfl.ch/) The SNPs were identified with SNP tool of the same ‘HTSstation’.The SNPs unique/over-represented in plus-phenotype pool compared to minus-phenotype pool were identified in Excel. The mutations in polymerase genes were confirmed by segregation of phenotype in back-crosses with strains harboring C-terminally tagged versions of polymerase genes and by Sanger sequencing of the mutation site.

FACS

FACS assays were performed essentially as in (Mattarocci et al., 2014).

PAGE-WB

126 PAGE-WB assays were performed essentially as in (Shyian et al., 2016).

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131 DISCUSSION

DNA replication initiation control by conserved protein Rif1

Origin firing starts from DDK-dependent phosphorylation of inactive Mcm2-7 double hexamer and recruitment of origin firing factors Sld3/7 and Cdc45 (Bell and Labib, 2016). Rif1 appears to act exactly at this DDK-controlled step of DNA replication initiation. Genetically, loss of RIF1 compensates for the deficiencies of DDK components (ts alleles of cdc7 and dbf4) and origin firing factors ‘reading’ Mcm2-7 phosphorylation (sld3, cdc45, dpb11).

Biochemically, absence of Rif1 leads to increase in Mcm4 and Sld3 phosphorylation.

Mechanistically, Rif1 recruits PP1 phosphatase, which in turn wipes off the phosphate residues from firing factors Mcm4 and Sld3. Several important questions to this model of Rif1 action remain. First, what is the complete set of firing factors regulated by DDK and Rif1-Glc7? Second, is Rif1 acting in cis at regulated origins by recruiting PP1 there or in trans (e.g.

by influencing limiting firing factors distribution due to competition of early, late and rARS origins)?

Rif1-PP1 targets relevant for DNA replication

Which proteins harbor the phosphosites relevant for Rif1-PP1 dependent DNA replication initiation regulation?

By candidate approach, we and others (Dave et al., 2014; Hiraga et al., 2014;

Mattarocci et al., 2014) have discovered that out of the pre-replicative complex specifically the Mcm4 subunit of the Mcm2-7 complex is regulated through Rif-PP1 dependent dephosphorylation. A recent phosphoproteomic analysis of chromatin fraction in HEK293 cells treated with siRNA targeting RIF1 also found that Mcm4 is the most hyperphosphorylated in this condition (Hiraga et al., 2017). Additionally, this study found that some residues in Mcm2 and Mcm6 are also more phosphorylated upon depletion of RIF1, albeit to lower extent.

Moreover, while searching for Rif1-PP1 targets, we observed that Sld3 protein in cells lacking RIF1 exhibit a slower-migrating set of bands (Mattarocci et al., 2014). These bands appeared in G1 arrested cells in DDK-dependent manner, persisted in early S phase and

132 disappeared in later S phase and we presumed these Sld3 protein species to be phosphorylated forms. These observations are consistent with recent report describing a specific interaction between DDK regulatory subunit Dbf4 and Sld3 (Fang et al., 2017).

Interestingly, the mcm4-NΔ allele of Mcm4 lacking the replication initiation inhibitory domain (Sheu and Stillman, 2010) has additive effect with rif1Δ mutation on the temperature sensitive growth of cdc7-4 cells (Stefano Mattarocci, personal communication), highlighting a possibility of additional relevant DDK-dependent sites in Mcm4, involvement of Sld3 phosphosites, other targets of DDK or altogether DDK-independent additional effect of RIF1 deletion in this case. Further deep phosphoproteomic investigation of cells lacking Rif1 in various physiological conditions should help to clarify this question.

Molecular mechanism of selectivity in Rif1 action at origins

Chromatin immunoprecipitation methods in budding yeast detect Rif1 only on telomeres and silent mating type loci (HML and HMR), where it is recruited through Rif1 C-terminus via interaction with Rap1 (Smith et al., 2003). Curiously, in mouse embryonic stem cells Rif1 could be detected at megabase-sized domains throughout chromosomes (Foti et al., 2016). Moreover, these Rif1 associated sites in mouse overlap with late-replicating domains, suggesting that Rif1 could indeed act in cis at the affected origins. Recently, our laboratory used a different method, chromosome endogenous cleavage (ChEC-seq), to detect regions of Rif1 association with chromosomes in budding yeast (Hafner et al., 2018).

This method allowed detecting Rif1 association at the late origins regulated by Rif1, including rARS at the rDNA locus.

How does Rif1 recognize the origins then? The N-terminal HEAT repeats of Rif1 is an avid DNA-binding module (Mattarocci et al., 2017). However, budding yeast Rif1 doesn’t appear to show sequence-specific binding to DNA (Mattarocci et al., 2017), although the ARS sequence was not assessed in this study. Masai laboratory presented several reports for preferential binding by fission yeast Rif1 to G-quadruplex (G4) forming DNA sequences (Kanoh et al., 2015) both in vitro and in vivo. These sequences were adjacent to Rif1-regulated dormant or late origins in vivo and mutation of some of them led to enhanced origin firing in a big domain in the vicinity of the cite (Kanoh et al., 2015). A more recent report confirmed that murine Rif1 is also capable of G4 sequence binding and bridging (Moriyama et al., 2018). It is conceivable therefore that Rif1 might either access origins by

133 binding to G4 sequences in their vicinity in cis or by affecting the genome architecture and thereby origin clustering and accessibility to firing factors in trans.

Alternatively, some structural or topological transitions at origin DNA during its activation could recruit Rif1. Indeed, Rif1 has preference in binding toward 3’ ssDNA junctions with dsDNA (Mattarocci et al., 2017) and ssDNA or ssDNA-dsDNA junctions could be formed during initial origin activation. Going further in this line of thoughts, we could imagine that initial origin melting at the earliest steps of origin firing could create a favorable substrate for Rif1-Glc7 recruitment and reversal of the activation. However, once the DDK-CDK activities overcome some threshold, their activation of origins would win over the inhibitory reversing activity of Rif1-Glc7. Still alternatively, protein factors binding to the origins could be responsible for Rif1 recruitment. For instance, as Rif1 interacts with Dbf4 component of DDK (Mattarocci et al., 2014), it could be expected to follow DDK at the sites of its localization at chromatin. Further studies will undoubtedly help to resolve which (if any) of these models is/are correct.

It is worth noting that one should not necessarily expect a direct interaction of Rif1 with all the origins it regulates. Indeed, the replication initiation limiting factors model predicts that a change in firing of a set of origins could lead to concomitant change of an opposite sign in firing of another set. This was proven to be the case with Sir2 and Rpd3 effects on origin firing throughout the genome that are mostly secondary to the primary effect of these two factors on the rDNA origins (rARS) (Yoshida et al., 2014). Indeed, deletion or knockdown of RIF1 leads not only to advancement of late origins, but also to a delay in firing of some of the earlies origins. For instance, centromere-proximal origins in budding yeast are delayed in activation in rif1Δ (Peace et al., 2014). It would be of interest then to see how many of the Rif1-dependent changes in replication timing are direct effects due to Rif1 binding and PP1 recruitment and how many are secondary downstream effects due to firing factors re-distribution. We imagine that rDNA locus may play a significant role in this interplay, as it contains a lion’s share of all potential origins in yeast genome and would be expected therefore to contribute to the competition. Specifically, we imagine that one of the biological roles of Rif1 in DNA replication and genome integrity could be to spread the firing of rDNA origins over a broad time period in the middle S phase. Yeast cell starts to duplicate genome from early origins (especially centromeric, where DDK level is high in G1, which is necessary to maintain genome integrity (Natsume et al., 2013) followed by rARS and then

134 late origins (and even dormant in case of replication delay). Longer rDNA replication could therefore be partially responsible for the separation in time of early and late origins. A cell lacking Rif1 would have higher DDK-dependent pre-RC priming at both rARS and late/dormant origins. This, in turn, would lead to advancement of rARS and late origins firing time and overall more stochastic firing of all origins (i.e. loss of spatiotemporal control).

Rif1 is a phosphoprotein regulated by CDK-DDK, Mec1-Rad53 and Glc7

Rif1 phosphoregulation by CDK-DDK, Mec1-Rad53 and Glc7 could ensure an ON-OFF switch mechanism for Rif1-Glc7 interaction. In this model, Glc7 association with Rif1 is

Rif1 phosphoregulation by CDK-DDK, Mec1-Rad53 and Glc7 could ensure an ON-OFF switch mechanism for Rif1-Glc7 interaction. In this model, Glc7 association with Rif1 is