Dbf4 Zn-Finger Motif Is Specifically Required for Stimulation of Ctf19-Activated Origins in Saccharomyces cerevisiae

Abstract

Eukaryotic genomes are replicated in spatiotemporal patterns that are stereotypical for individual genomes and developmental profiles. In the model system Saccharomyces cerevisiae, two primary mechanisms determine the preferential activation of replication origins during early S phase, thereby largely defining the consequent replication profiles of these cells. Both mechanisms are thought to act through specific recruitment of a rate-limiting initiation factor, Dbf4-dependent kinase (DDK), to a subset of licensed replication origins. Fkh1/2 is responsible for stimulation of most early-firing origins, except for centromere (CEN)-proximal origins that recruit DDK via the kinetochore protein Ctf19, which is required for their early firing. The C-terminus of Dbf4 has been implicated in its recruitment to origins via both the Fkh1/2 and Ctf19 mechanisms. Here, we show that the Zn-finger motif within the C-terminus is specifically required for Dbf4 recruitment to CENs to stimulate CEN-proximal/Ctf19-dependent origins, whereas stimulation of origins via the Fkh1/2 pathway remains largely intact. These findings re-open the question of exactly how Fkh1/2 and DDK act together to stimulate replication origin initiation.

Keywords: Ctf19; Dbf4-dependent-kinase; Fkh1; chromosome domains; replication origins.

Figure 1

Dbf4∆C is defective in essential Dbf4 function(s). (A) Schematic representation of Dbf4 protein domains with sequence detail of the Zn-finger domain; amino acid changes in the Zn* alleles are indicated in lower case, red font. (B) Strains CVy63 (WT), HYy176 (dbf4ΔC), MPy74 (dbf4-Zn*1), MPy76 (dbf4-Zn*2), MPy86 (dbf4-Zn*1 fkh1∆), MPy90 (dbf4-Zn*2 fkh1∆), HYy210 (cft19∆), HYy211 (cft19∆ dbf4∆C), HYy207 (cft19∆ fkh1Δ fkh2-dsm), and HYy218 (fkh1Δ fkh2-dsm) were grown to mid-log phase, diluted, and plated onto rich media, then incubated at the indicated temperatures and imaged after 2–3 days.

Figure 2

Dbf4∆C is defective in bulk genome replication, while Dbf4-Zn* mutants are not. Strains described in Figure legend 1 plus HYy217 (fkh1∆) and HYy215 (dbf4∆C—instead of HYy176) were synchronized in G1 phase and released into S phase for DNA content analysis. Note the different time intervals of the different panels, and the unique time intervals for dbf4∆C (inset). Vertical red lines indicate 1C (un-replicated) and 2C (replicated) chromosomal DNA contents, respectively.

Figure 3

Dbf4-Zn* is defective in CEN-proximal origin firing. Strains described in Figure 1 and Figure 2 legends were synchronized in G1 phase, released into S phase with HU for 60 min (90 min for dbf4∆C), and harvested for QBU analysis. (A) QBU values averaged for two replicates and scale-normalized across strains are shown for representative chromosome XV; origins and origin sub-groups are indicated with color-coded circles below the x-axis. (B) Two-dimensional scatter plots comparing QBU signals averaged across 500 bp regions centered on 410 confirmed origins; origins and sub-groups are color-coded as indicated. (C) Boxplot distributions of averaged QBU counts across 500 bp regions aligned on origins of the indicated groups; the number of origins in each group is indicated within parentheses; results of statistical analyses are given in Table S1. (D) Two-dimensional scatter plot comparing the differences in QBU values for ctf19∆ (WT-ctf19∆) versus dbf4-Zn* (WT-dbf4-Zn*) for Fkh-activated and CEN-proximal origins.

Figure 4

Dbf4-Zn* retains Fkh1 origin stimulation. Strains described in Figure 1 and Figure 2 legends were synchronized in G1 phase, released into S phase with HU for 60 min, and harvested for QBU analysis. (A) QBU values averaged for two replicates and scale-normalized across strains are shown for representative chromosome XV; origins and origin sub-groups are indicated with color-coded circles below the x-axis. (B) Two-dimensional scatter plots comparing QBU signals averaged across 500 bp regions centered on 410 confirmed origins; origins and sub-groups are color-coded as indicated. (C) Boxplot distributions of averaged QBU counts across 500 bp regions aligned on origins of the indicated groups; the number of origins in each group is indicated within parentheses; results of statistical analyses are given in Table S1.

Figure 5

Dbf4-Zn* is defective in its recruitment to CENs. Strains EAy1 (3xFLAG-DBF4) and EAy2 (3xFLAG-dbf4-Zn*1) were synchronized in G1 phase and subjected to ChIP-seq analysis. (A) Heatmaps of averaged ChIP-seq values across 10kbp regions centered on origins (n = 408 because two rDNA origins removed), origin sub-groups, or CENs. (B) Boxplot distributions of ChIP-seq values for 500 bp windows centered on the indicated features, subjected to two-sided t-tests. (C) Two-dimensional scatter plots of ChIP-seq values for 500 bp windows centered on the indicated features, showing lines of best fit for origin sub-groups and CENs.