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Mol Biol Cell. Dec 2008; 19(12): 5550–5558.
PMCID: PMC2592646
An InCytes from MBC Selection

The Hsk1(Cdc7) Replication Kinase Regulates Origin Efficiency

Daniel J. Lew, Monitoring Editor

Abstract

Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or decreasing Hsk1-Dfp1 levels correspondingly increases or decreases origin efficiency. Furthermore, tethering Hsk1-Dfp1 near an origin increases the efficiency of that origin, suggesting that the effective local concentration of Hsk1-Dfp1 regulates origin firing. Using photobleaching, we show that Hsk1-Dfp1 is freely diffusible in the nucleus. These results support a model in which the accessibility of replication origins to Hsk1-Dfp1 regulates origin efficiency and provides a potential mechanistic link between chromatin structure and replication timing. By manipulating Hsk1-Dfp1 levels, we show that increasing or decreasing origin firing rates leads to an increase in genomic instability, demonstrating the biological importance of appropriate origin efficiency.

INTRODUCTION

Eukaryotic DNA replication initiates at discrete replication origins. The mechanisms by which individual origins are established during G1 and fired during S phase are understood in some detail (Sclafani and Holzen, 2007 blue right-pointing triangle). However, many questions about the regulation and coordination of origin firing remain (Gilbert, 2001 blue right-pointing triangle). In particular, it is not understood why, of the many origins that are established during G1, only a subset fire in each S phase. In fission yeast and vertebrates, <50% of potential origins actually fire in each S phase (Dijkwel et al., 2002 blue right-pointing triangle; Jeon et al., 2005 blue right-pointing triangle; Feng et al., 2006 blue right-pointing triangle; Heichinger et al., 2006 blue right-pointing triangle; Lebofsky et al., 2006 blue right-pointing triangle; Patel et al., 2006 blue right-pointing triangle; Eshaghi et al., 2007 blue right-pointing triangle; Mickle et al., 2007 blue right-pointing triangle). This inefficient origin firing leads to a different subset of origins firing in each cell. Origins that do not fire are passively replicated by replication forks from neighboring origins, but they will fire if passive replication is prevented, suggesting that they are functional (Santocanale et al., 1999 blue right-pointing triangle; Vujcic et al., 1999 blue right-pointing triangle; Kim and Huberman, 2001 blue right-pointing triangle). The excess origins may serve as backups to ensure efficient replication if origin firing is compromised or replication forks stall (Hyrien et al., 2003 blue right-pointing triangle; Legouras et al., 2006 blue right-pointing triangle; Woodward et al., 2006 blue right-pointing triangle; Ge et al., 2007 blue right-pointing triangle). The importance of maintaining normal levels of origin efficiency is suggested by the fact that both increasing and decreasing origin efficiency correlates with decreased genomic stability (Spruck et al., 1999 blue right-pointing triangle; Lengronne and Schwob, 2002 blue right-pointing triangle).

The origin activation cycle can be divided into four stages (Sclafani and Holzen, 2007 blue right-pointing triangle). First, the origin recognition complex (ORC) binds to the origin DNA. In the second stage, referred to as licensing, ORC, in collaboration with Cdc6 and Cdt1, loads the minichromosome maintenance (MCM) complex to establish a prereplication complex (pre-RC). In the third stage, during S phase, pre-RCs are activated by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) phosphorylation, which allows recruitment of additional factors, including Cdc45 and the GINS complex. Finally, the active origin is unwound, and the replication forks are established. Firing of an origin, or passive replication by a replication fork from a neighboring origin, disassembles the pre-RC and resets the cycle. The regulation of origin efficiency could occur at any of these steps in origin establishment and activation. However, because excess origins have established, functional pre-RCs, it seems that regulation occurs after the licensing step (Shirahige et al., 1998 blue right-pointing triangle; Santocanale et al., 1999 blue right-pointing triangle; Vujcic et al., 1999 blue right-pointing triangle; Kim and Huberman, 2001 blue right-pointing triangle; Hayashi et al., 2007 blue right-pointing triangle).

We have recently shown that origin firing in fission yeast is stochastic (Patel et al., 2006 blue right-pointing triangle). That is, origins fire independently of their neighbors. We speculate that the same rate-limiting step underlies both the stochastic and inefficient qualities of origin firing. If the step that limits the efficiency of origin firing is randomly distributed among all potential origins, it will lead to the observed stochastic firing of origins. A plausible candidate for a rate-limiting step is origin phosphorylation by a freely diffusible kinase. Such a kinase would be expected to be required for origin firing throughout S phase, as is DDK (Bousset and Diffley, 1998 blue right-pointing triangle; Donaldson et al., 1998 blue right-pointing triangle). We show here that the Hsk1-Dfp1 kinase, the fission yeast DDK, is a freely diffusible, rate-limiting activator of origin firing.

MATERIALS AND METHODS

The strains used in this study are listed in Table 1. They were made and maintained using standard methods (Forsburg and Rhind, 2006 blue right-pointing triangle). Unless otherwise stated, cells were grown at 25°C in yeast extract plus supplements (YES).

Table 1.
Strains used in this study

Strain and Plasmid Construction

The primers used for making strains and plasmids are listed in Supplemental Table 1. The adh1:dfp1 allele was made in yFS240 by one-step promoter replacement using pFS255 and primers P96 and P97 (Sivakumar et al., 2004 blue right-pointing triangle). The GAL4-dfp1-3HA allele was constructed by amplifying the dfp1 open reading frame (ORF) from genomic DNA by using P112 and P113 and cloning in into SalI, XmaI-cut pFA6a-3HA-kanMX6 to create pFS295 (Wach et al., 1994 blue right-pointing triangle). The coding sequence for the GAL4 DNA binding domain (codons 1-147) was amplified from Saccharomyces cerevisiae genomic DNA by using P156 and P157 and cloned into BsiWI, AvrII-cut pFS295 to make pFS296. The fusion ORF was then amplified from pFS296 with P166 and P167 and transformed into yFS240 to replace genomic dfp1 ORF. The 5x Gal4 binding site cassette was amplified from pFS294 and integrated into the Gal4-Dfp1 strain by using P127 and P128 for integration near AT3003 and P138 and P139 for integration near AT2103 to create yFS459 and yFS460, respectively. pFS294 was created by removing the 5x Gal4 DNA binding site cassette from pDZ43 with BglII and SalI and cloning it into a variant of pFA6a-kanMX6, which had been cut with HpaI and EcoRV and religated (Wach et al., 1994 blue right-pointing triangle; Zappulla et al., 2002 blue right-pointing triangle). The strain yFS449 expressing Dfp1–2xGFP was made by sequentially integrating green fluorescent protein (GFP) as a C-terminal tag of Dfp1 in yFS104, first from pFA6a-GFP(S65T)-hphMX6 by using P213 and P167 and then from pFA6a-GFP(S65T)-natMX6 by using P129 and P216 (Sato et al., 2005 blue right-pointing triangle). pFS260 contains ura4 and the ars1 fragment from pFS253 cloned into pLIT28 (New England Biolabs, Ispwich, MA) (Sivakumar et al., 2004 blue right-pointing triangle).

Statistical Analyses

For comparisons of origin efficiency and for correlation between the firing of neighboring origins, p is calculated from Fisher's exact test. In the former case, p < 0.05 indicates a significant difference between experiment and control; in the latter case, p > 0.05 indicates a lack of correlation. For curve fitting, R2 is calculated from a linear fit on a semi-log plot; p is calculated from the χ2 statistic for the fit, weighted with the SD of each point; p > 0.05 indicates a good fit.

Origin Efficiency Assays

Cell synchronization, labeling, DNA combing, and fluorescence in situ hybridization (FISH) were performed as described previously (Patel et al., 2006 blue right-pointing triangle). Briefly, elutriation-synchronized, G2 cultures were grown in YES with 12 mM hydroxyurea (HU) and 500 nM 5-bromo-2′-deoxyuridine (BrdU) for 4 h to allow cells to complete their first G2, M, and G1 phases, and arrest in S phase. Cells were harvested and set in agarose plugs in which genomic DNA was prepared by SDS/protease-K digestion. The plugs were melted and digested with β-agarase to produce high-molecular-weight DNA in solution. The DNA was combed by dipping and slowly withdrawing a specially silanized glass coverslip, drawing out DNA molecules that have bound nonspecifically to the glass by an end, and laying the stretched DNA flat against the surface. The combed molecules were visualized by epifluorescence microscopy with anti-DNA and anti-BrdU antibodies. The cosmids C25H2, C3B8, and C2G2 were used for FISH probes in the ars727 region.

Fluorescence Redistribution after Photobleaching (FRAP) and Fluorescence Loss in Photobleaching (FLIP) Analysis

Dfp1-2xGFP (yFS449) cells were grown to mid-log at room temperature in YES, concentrated by centrifugation, and mounted in YES on a glass slide under a coverslip affixed by surface tension. Cells were imaged on a Leica SP2 laser scanning confocal microscope. Cells were imaged at 15% laser power to reduce bleaching during imaging. For FRAP, we used the FRAP application module and bleached a rectangular region of interest constituting the central one quarter of the nucleus at 100% laser power. For FLIP, we used the spot-bleach function at 100% laser power. For each experiment, we bleached one nucleus in a septated cell; the other was use as a control for bleaching during imaging. Images were saved as TIFF files and analyzed in ImageJ (National Institutes of Health, Bethesda, MD). All measurements were background corrected, and the change in signal from the bleach nucleus was normalized for bleaching during imaging. Images were uniformly adjusted for contrast, brightness, and pixel input range by using Canvas (Deneba Systems, Miami, FL). The graph of postbleach fluorescence versus bleach time was fit to the single exponential y = 0.96 × e^(−x/78), with R2 = 0.98, p = 0.37 in Igor Pro6 (Wavemetrics, Lake Oswego, OR), yielding τ = 78 ± 7 ms. We approximated the effective diffusion coefficient D from the radius of the nucleus Rn (~1.1 μm), the radius of the bleach volume Rb (~0.4 μm), and τ (~78 ms), with the equation D ≈ (Rn − Rb)2/τ ≈ 6 μm2/s. We determined the geometry of the bleach volume by bleaching histone 2B-GFP cells fixed in 70% ethanol. The z-stack reconstructions of cells bleached for 50 ms showed the laser bleaches a roughly cylindrical volume, with a diameter of ~0.8 μm. Numerical modeling suggests that systematic errors in our measurements will affect our results by no >50%, giving a confidence interval for the effective diffusion coefficient from 4 to 10 μm2/s.

Genome Stability Assays

Wild-type (yFS240), adh1-dfp1 (yFS458) and hsk1-1312 (yFS457) strains were transformed with either pFS260 or pDblet (Brun et al., 1995 blue right-pointing triangle). The transformants were grown in a selective medium containing leucine, adenine and histidine (LAH) until mid-log phase (~OD 2), diluted 40-fold into nonselective YES, and repeatedly diluted whenever the cultures reached mid-log phase. At 0, 6, 16, 24, 48, 72, 96, and 124 h in YES, the cell density was counted, and equal numbers of cells were plated on YES and LAH. After 5–7 d, the numbers of colonies were counted on each plate. The experiment was performed three times. The rate of plasmid loss per generation, L, was calculated from the ratio of the number of colonies on selective versus nonselective plates for a particular strain by using the equation L = 100(1 − ex), where x = (ln(Rn/R0))/n (Huberman, 1999 blue right-pointing triangle). R0 is the proportion of cells retaining plasmid at the start in nonselective media; Rn is the proportion of cells retaining plasmid after n generations in nonselective media.

For minichromosome loss assays, yFS461, yFS462, yFS463, and yFS464 strains carrying the Ch16 minichromosome were grown in a defined medium containing leucine, uracil and histidine until mid-log phase and then diluted into YES with or without 1 mM HU. Similar to the plasmid loss assay described above, whenever the cells reached to mid-log phase they were diluted 40-fold with YES. After ~50 generations in YES, cells were counted and plated on YEA, which is low in adenine and causes Ade− colonies to become pink. After 5–7 d at 30°C, the total numbers of white and pink colonies were counted. Loss rates were calculated as described above.

RESULTS

Hsk1-Dfp1 Is Rate Determining for Origin Firing

If origin phosphorylation by the Hsk1-Dfp1 kinase is a stochastic event that leads to random origin activation, then raising or lowering the activity or abundance of Hsk1-Dfp1 should concomitantly increase or decrease the efficiency of origin firing. To test this prediction, we measured the efficiency of origin firing in early S phase after manipulating the activity of Hsk1-Dfp1.

To reduce the activity of the Hsk1-Dfp1 kinase, we used the temperature-sensitive hsk1-1312ts allele. Because hsk1 is essential and because hypomorphic alleles have pleiotropic phenotypes, we examined hsk1-1312ts cells for a temperature at which Hsk1-Dfp1 activity would be only mildly decreased, so that we might see an effect on origin firing efficiency without disrupting replication altogether (Snaith et al., 2000 blue right-pointing triangle). We found that 25°C is the highest temperature at which hsk1-1312 cells grow well and show none of the previously reported pleiotropic phenotypes. To assay the rate of origin firing, we visualized active origins, which had been labeled in vivo with BrdU, on individual DNA molecules (Patel et al., 2006 blue right-pointing triangle). Cells were elutriation synchronized in G2 and followed through mitosis and into S phase in the presence of the BrdU label and HU, which prevents deoxynucleotide synthesis and arrests cells in early S phase. This protocol allows origins to fire in early S phase and for BrdU to be incorporated in the newly synthesized DNA around those origins. Then, as nucleotides are depleted by replication, forks stall and the replication checkpoint inhibits further origin firing (Santocanale and Diffley, 1998 blue right-pointing triangle). The labeled DNA molecules are prepared by DNA combing, in which individual, protein-free DNA fibers are stretched on a glass surface and visualized by epifluorescence microscopy (Bensimon et al., 1994 blue right-pointing triangle). BrdU incorporation marks the origins on those fibers that fired before depletion of nucleotides caused replication to arrest.

We examined the effect of reducing Hsk1-Dfp1 activity on the efficiency of the well-studied origins in the ura4 region of chromosome III (Dubey et al., 1994 blue right-pointing triangle). To identify these origins, we combined DNA combing with fluorescent in situ hybridization (Figure 1A and Supplemental Figure S1A). This technique allows us to identify fibers from specific regions of the genome and to measure the efficiency of specific origins in those regions. On 67 fibers, we found that the efficiency of five origins from the ura4 region was reduced by 49%, on average (the range is from 39 to 53%; Figure 1, B and C). By Fisher's exact test of the aggregate data, the difference is highly significant (p = 7.3 × 10−8).

Figure 1.
Hsk1-Dfp1 is rate determining for origin firing. (A) The ura4 region of chromosome III and representative combed molecules. The graphic represents the left end of chromosome III. Blue represents coding sequences, black noncoding sequences, green noncoding ...

To quantitate the effect of a reduction of Hsk1-Dfp1 activity on origin efficiency across the genome, we measured the distances between origins on unselected combed fibers. In 146 measurements, the average interorigin distance is 64% longer than the wild-type data set (149 vs. 91 kb). In addition, the size of the stalled replicons increase 2.4-fold (median size, 23.2 vs. 9.7 kb), as would be expected if there are fewer forks to deplete the available deoxynucleotides. These results suggest that fewer origins fire across the genome and corroborate the reduction in efficiency of the origins in the ura4 region.

To increase the activity of the Hsk1-Dfp1 kinase, we overexpressed Dfp1 from the constitutive adh1 promoter, leading to an approximately threefold increase in Dfp1 protein and Hsk1-Dfp1 kinase activity relative to wild-type S phase levels (Supplemental Figure S2). The adh1:dfp1 cells grow normally and have normal bulk replication kinetics by flow cytometry. However, the increase in Hsk1-Dfp1 kinase activity leads to an increase in origin firing efficiency. On 54 fibers, we found that the efficiency of five origins from the ura4 region increased by 61%, on average (the range is from 20 to 136%; p = 8.0 × 10−7; Figure 1, B and C). In 443 whole-genome measurements, the average interorigin distance is 24% less than wild type (69 vs. 91 kb) and the replicon size was 20% smaller (median size, 7.8 vs. 9.7 kb), consistent with the increased efficiency seen in the ura4 region.

In wild-type fission yeast, origin firing is stochastic (Patel et al., 2006 blue right-pointing triangle). To determine whether it is still stochastic in these strains, we measured the correlation between the firing of neighboring origins in the ura4 region. Similar to wild type, we see no significant positive or negative correlation between the firing of neighboring origins (p > 0.1).

Hsk1-Dfp1 Acts in cis to Regulate Origin Firing

Hsk1 and its homologues are known to bind to pre-RCs and phosphorylate the MCM complex (Pasero et al., 1999 blue right-pointing triangle; Duncker et al., 2002 blue right-pointing triangle; Masai et al., 2006 blue right-pointing triangle; Sheu and Stillman, 2006 blue right-pointing triangle). These observations, together with our results demonstrating that Hsk1-Dfp1 is a rate-determining factor for origin firing, suggest that Hsk1-Dfp1 phosphorylation of origins regulates their efficiency. However, it is also possible that Hsk1-Dfp1 indirectly regulates origin efficiency through other targets. For example, in budding yeast overexpression of Dbf4 interferes with the replication checkpoint, which could in turn affect origin efficiency. To test whether overexpression of Dfp1 interferes with or activities the replication checkpoint in fission yeast, we assayed HU sensitivity and Cds1 kinase activity in adh1:dfp1 cell. We find no evidence of HU sensitivity, Cds1 inhibition or Cds1 activation, suggesting that the effects of Dfp1 overexpression are not due to indirect effects on the replication checkpoint (Supplemental Figure S3).

To directly test the hypothesis that Hsk1-Dfp1 acts directly on origins, we tethered it to a specific locus via a Gal4-Dfp1 fusion. We reasoned that if Hsk1-Dfp1 acts in cis, then increasing its local concentration at a locus should increase the efficiency of origins near that locus. We fused the DNA binding domain of Gal4 (amino acids 1–147) to the amino terminus of Dfp1 at its genomic locus; the strain expressing the Gal4-Dfp1 allele is wild type by standard growth and viability assays. We then integrated five Gal4 binding sites near the ura4 locus, between the moderately efficient origin AT3003 and the inefficient origin AT3002.8. We examined 250 origin firings in the Gal4-Dfp1 strain—123 on 47 fibers with the Gal4 binding sites and 127 on 63 fibers without Gal4 binding sites—by using the HU-block/FISH approach described above. Consistent with our model, we see a 3.6-fold increase in efficiency of AT3002.8 (from 12 to 43%; p = 2.8 × 10−4 as measured by Fisher's exact test) and a 1.6-fold increase in the efficiency of AT3003 (from 42 to 66%; p = 1.1 × 10−2), but no significant effect on the flanking origins AT3002 and AT3004 (Figure 2A and Supplemental S1B). These results demonstrate that Hsk1-Dfp1 is rate determining in cis and suggest that the rate-determining step is direct phosphorylation of the origin by the Hsk1-Dfp1 kinase.

Figure 2.
Hsk1-Dfp1 acts in cis to regulate origin firing. (A) 5 Gal4 DNA-binding sites were integrated adjacent to origin AT3003. The sites are ~3 kb from AT3003, 6 kb from 3002.8, and 40 kb from both AT3002 and AT3004. A map of the region is shown below ...

Most identified fission yeast origins fire with between 20 and 50% efficiency (Heichinger et al., 2006 blue right-pointing triangle; Patel et al., 2006 blue right-pointing triangle; Feng et al., 2006 blue right-pointing triangle; Mickle et al., 2007 blue right-pointing triangle). However, rare origins have been identified that fire very inefficiently, if at all, but do have assembled pre-RCs (Kim and Huberman, 2001 blue right-pointing triangle; Feng et al., 2006 blue right-pointing triangle; Hayashi et al., 2007 blue right-pointing triangle). To test whether such cryptic origins are also limited by Hsk1-Dfp1 activity, we tethered Gal4-Dfp1 near the well-studied, cryptic origin ars727, which is called AT2103 in the systematic nomenclature of Segurado et al. (Kim and Huberman, 2001 blue right-pointing triangle; Segurado et al., 2003 blue right-pointing triangle; Yompakdee et al., 2004 blue right-pointing triangle). We examined 125 origins firings—74 on 53 fibers with the Gal4 binding sites and 51 on 60 fibers without Gal4 binding sites—and we found that local tethering of Gal4-Dfp1 increases the efficiency of AT2103 3.3-fold (from 6 to 20%; p = 2.2 × 10−2) and increases the efficiency of the neighboring origin AT2102 from 0 to 17% (p = 1.2 × 10−3) without significantly affecting other origins in the region (Figure 2B and Supplemental S1B). These results show that even extremely inefficient origins can be activated by Hsk1-Dfp1, suggesting that Hsk1-Dfp1 is rate limiting for all origins, not just more efficient origins.

Although the simplest explanation for the effect of tethering Hsk1-Dfp1 on the efficiency of local origins in that Hsk1-Dfp1 is directly activating the origins by phosphorylating MCM, it is also possible that the local high concentration of Hsk1-Dfp1 affects local chromatin structure, which in turn indirectly affects local origin efficiency. We reasoned that any effect on local chromatin structure that would affect origin efficiency also would affect transcription. Therefore, to test for local chromatin affects, we used genome-wide transcriptional profiling to assay transcript levels in cells with and without Hsk1-Dfp1 tethered near the ura4 locus (Oliva et al., 2005 blue right-pointing triangle). We find no significant difference in transcript levels near ura4 between wild-type cells and cells with Gal4-Dfp1 tethered at AT3003 (Supplemental Figure S4). Specifically, the change in transcript levels for the 24 genes within the 50 kb around AT3003 is 1.02-fold, compared with a genome-wide change of 1.01-fold (p > 0.2). These results suggest that the increased local concentration of Hsk1-Dfp1 is not affecting origin efficiency indirectly through local chromatin effects.

Hsk1-Dfp1 Is Freely Diffusible in the Nucleus

Our model that Hsk1-Dfp1 phosphorylation leads to the stochastic firing of origins predicts that Hsk1-Dfp1 should be freely diffusible in the nucleus and able to randomly interact with all potential origins. To test this prediction, we tagged Dfp1 with two copies of GFP and performed FRAP and FLIP experiments. The Dfp1-2xGFP strain grows normally, has wild-type replication kinetics by flow cytometry, and expresses approximately wild-type levels of the Dfp1 fusion protein (Supplemental Figure S2). As expected, Dfp1-GFP was localized primarily to the nucleus. For all of our photobleaching experiments, we use septated cells, which serve in fission yeast as a marker for S-phase cells. For FRAP experiments, we bleached a rectangular strip constituting the central quarter of the nucleus using the laser from a confocal microscope. In traditional FRAP analysis, one measures the kinetics of the redistribution of unbleached fluorescent molecules and from that data extracts the effective diffusion coefficient. However, in the 0.35 s that it took to image the nucleus after bleaching, the unbleached Dfp1-GFP uniformly redistributed throughout the nucleus. Because the fluorescence redistribution is faster than we can measure, FRAP only allows us to put a lower limit on the diffusibility of Hsk1-Dfp1. Using a standard FRAP model, we estimate that the effective diffusion coefficient for Dfp1-GFP is > 0.5 μm2/s (Axelrod et al., 1976 blue right-pointing triangle; Carrero et al., 2003 blue right-pointing triangle).

To estimate more accurately the effective diffusion coefficient, we developed a novel variant of the FLIP approach, which allows for the measurement of high effective diffusion coefficients without the need for fast data acquisition. In this technique, we bleach a small volume in the nucleus for varying times. When the bleach pulse is very short, only the molecules in the bleach volume are bleached, and the overall fluorescence of the nucleus is reduced by an amount proportional to the volume of the bleach spot. However, when the bleach pulse is longer, molecules have time to diffuse in and out of the bleach volume. Thus, more molecules are bleached, and the overall fluorescence of the nucleus is further reduced. From the graph of bleach time versus loss of fluorescence, we can extract the effective diffusion constant. The major advantage of this approach is that it is limited by the duration of the bleach pulse, which can easily be on the millisecond time scale, not the speed of imaging the sample, which can take hundreds of milliseconds.

We bleached an ~0.8-μm-diameter spot—constituting about one fifth the volume of the 2.3-μm nucleus—for various times (Figure 3). When we bleached for 300 ms, 97% of the GFP signal was bleached, demonstrating that the bulk of the Dfp1-GFP moved through the bleached volume within 300 ms (Figure 3B). Shorter length pulses bleached less of the Dfp1-GFP fluorescence. By fitting an exponential curve to the graph of fluorescence versus bleach time, we estimated the average time until bleaching for any single GFP molecule to be 79 ms, and from that we estimated an effective diffusion coefficient of 6 μm2/s (see Materials and Methods for details). For comparison, the freely diffusible mammalian nuclear proteins HMG-17 and fibrillarin have effective diffusion coefficients ~0.5 μm2/s (Phair and Misteli, 2000 blue right-pointing triangle). These diffusion coefficients are lower than the 20 μm2/s of free nuclear GFP, suggesting that the diffusion of these proteins is slowed by interaction with other proteins in the nucleus (Phair and Misteli, 2000 blue right-pointing triangle; Essers et al., 2002 blue right-pointing triangle). In Hsk1-Dfp1, effective diffusion could be slowed by its association with chromatin and, in particular, origins (Takeda et al., 1999 blue right-pointing triangle; Weinreich and Stillman, 1999 blue right-pointing triangle). However, the important conclusion is that Dfp1-GFP, and presumably Hsk1-Dfp1 complexes, diffuse rapidly in the nucleus on the 20-min time scale of replication. In particular, the average time it takes a molecule of Dfp1-GFP to bleach (~0.1 s) is approximately the time that it takes it to diffuse across the nucleus.

Figure 3.
Hsk1-Dfp1 is freely diffusible in the nucleus. (A) dfp1-GFP cells (yFS449) were imaged before and after bleaching for the indicated time with an SP2 laser scanning confocal microscope (Leica, Wetzlar, Germany). Bleaching was preformed with the spot bleach ...

Hsk1-Dfp1 Levels Affect Plasmid and Chromosome Stability

Because the levels of Hsk1-Dfp1 activity regulate the efficiency of replication origins, such regulation is likely to benefit cells, perhaps by increasing genomic stability. For example, a reduction of origin efficiency could decrease genome stability by increasing the distance between active origins and thus increasing the chances of replication fork collapse, whereas an increase in origin efficiency could tax nucleotide pools or repair processes, making fork stalls more common or less efficiently repaired. This hypothesis predicts that either raising or lowering Hsk1-Dfp1 activity should increase genome instability. To test these predictions, we assayed the stability of two reporters: a plasmid that contains a single origin and a minichromosome that contains many origins.

The plasmid-stability assay is an indirect measure of origin efficiency. Because the plasmid has only one origin and that origin must fire for the plasmid to be inherited by both daughters, the stability of the plasmids is dominated by the efficiency of the plasmid origin. As expected, using hsk1-ts to reduce Hsk1-Dfp1 activity results in an increase in the rate of plasmid loss by almost twofold (Figure 4A). Conversely, overexpressing Dfp1 decreases plasmid loss by almost fivefold. As a control to demonstrate that the effect we measured was primarily due to origin efficiency, we repeated the experiments with pDblet, a plasmid carrying two copies of ars3002 (Figure 4A) (Brun et al., 1995 blue right-pointing triangle). The extra origin makes the plasmid almost fivefold more stable in wild-type cells, an effect comparable with the overexpression of Dfp1. Furthermore, overexpression of Dfp1 does not significantly increase the stability of pDblet, suggesting that pDblet stability is not limited by origin efficiency. However, pDblet is almost threefold less stable in hsk1-ts cells, suggesting that the plasmid is still sensitive to reductions in origin efficiency.

Figure 4.
Hsk1-Dfp1 levels affect plasmid and chromosome stability. (A) Wild-type (yFS240), hsk1-ts (yFS457), and adh1:dfp1 (yFS458) strains carrying plasmids with one copy (pFS118) or two copies of ars3002 (pDblet) were analyzed for plasmid loss rates. The data ...

The minichromosome assay measures global genome stability because it does not depend on the firing of any one origin. Instead, minichromosome Ch16 contains many origins, and the effect of origin efficiency on its loss is likely to be due to indirect effects on replication fork stability. We measured the loss of Ch16 in wild-type and adh1:dfp1 cells and found no significant difference (Figure 4B). However, it is possible that any replication problems due to the increase in origin efficiency caused by overexpression of Dfp1 can be efficiently resolved by the replication checkpoint. We tested the importance of the replication checkpoint in maintaining the stability of Ch16 in adh1:dfp1 cells by deleting the cds1 checkpoint gene. Ch16 stability is reduced over 10-fold in adh1:dfp1 cds1Δ cells, significantly lower than in either adh1:dfp1 or cds1Δ cells alone, and lower even than the additive effect of the two genotypes (Figure 4B). This result demonstrates that the checkpoint is required to stabilize chromosomes in adh1:dfp1 cells and suggests that abnormally high origin firing efficiencies lead to increased replication fork density, requiring the activity of the checkpoint to prevent increased irreparable fork collapse. However, because Hsk1-Dfp1 is thought to be directly involved it fork metabolism, it is also possible that the increase Hsk1-Dfp1 activity could directly affect fork stability.

The hypothesis that increased replication fork density in adh1:dfp1 cells leads to increased requirement for the replication checkpoint predicts that chromosome stability in adh1:dfp1 cells should be sensitive to mild replication stress caused by a moderate reduction in nucleotide levels. To test this hypothesis, we induced replication stress by growing the cells in a low level of hydroxyurea. The addition of 1 mM HU did not decrease the stability of Ch16 in wild-type cells, but it did decrease the stability of Ch16 in adh1:dfp1 cells by more than threefold. This result suggests that the increased origin firing in adh1:dfp1 cells interferes with the ability of cells to cope with the replication stress caused by nucleotide depletion. To test whether this ability requires the replication checkpoint, we examined chromosome stability in HU-treated adh1:dfp1 cds1Δ cells. Ch16 stability under these conditions is reduced more than sevenfold, significantly more than in either adh1:dfp1 or cds1Δ cells alone, and more even than the additive effect of the two genotypes (Figure 4B). Together, the plasmid and minichromosome stability results show that either increasing or decreasing origin efficiency decreases genome stability and suggest that cells regulate the efficiency of their origins by setting the level of Hsk1-Dfp1 activity to maximize genome stability.

DISCUSSION

Replication origins in fission yeast fire inefficiently and stochastically (Dubey et al., 1994 blue right-pointing triangle; Feng et al., 2006 blue right-pointing triangle; Heichinger et al., 2006 blue right-pointing triangle; Patel et al., 2006 blue right-pointing triangle; Eshaghi et al., 2007 blue right-pointing triangle; Hayashi et al., 2007 blue right-pointing triangle; Mickle et al., 2007 blue right-pointing triangle). We reasoned that both the inefficient and stochastic nature of origin firing could be explained if the rate of origin activation was regulated by a freely diffusible, rate-limiting factor. Such a factor would randomly interact with and activate licensed replication origins, leading to stochastic activation. But, because it is limiting, it would be able to activate only a subset of the licensed origins in any given S phase, leading to inefficient firing. We further reasoned that if such a factor was overexpressed, it should lead to increased origin firing. We therefore took a candidate protein approach, considering proteins known to be required for pre-RC formation and activation, including Cdc18, Mcm10, Cdc45, and the Hsk1-Dfp1 replication kinase. We obtained positive results with Hsk1-Dfp1 before obtaining conclusive results for other candidates and have thus focused on Hsk1-Dfp1.

Our results show that increasing or decreasing Hsk1-Dfp1 kinase activity leads to an increase or decrease in origin firing efficiency, respectively (Figure 1). We measured origin efficiency by labeling the nascent DNA at active origins in vivo with BrdU and visualizing the BrdU incorporation patches on individual combed DNA molecules. In combination with FISH to identify fibers from specific regions of the genome, this approach allows us to directly count how often a specific origin fires in a population of cells. Because we can count large numbers of fibers, we can measure changes in origin efficiency as small as 20% with high statistical confidence. To facilitate the unambiguous identification of origins that fire early in S phase and to prevent the fusion of BrdU incorporation patches from neighboring origins, we analyzed synchronized cells in early S phase and performed our BrdU labeling the presence of HU. HU prevents deoxynucleotide synthesis and blocks replication after ~10% of the genome is replicated (Patel et al., 2006 blue right-pointing triangle). After origins have fired early in S phase, the early forks progress ~5 kb and in the process reduce deoxynucleotide levels to a point at which the replication checkpoint is triggered, forks stall, and further origin firing is prevented (Santocanale and Diffley, 1998 blue right-pointing triangle; Kim and Huberman, 2001 blue right-pointing triangle). Therefore, the checkpoint is not activated until after the firing of origins in early S phase, and thus the checkpoint is presumed not to affect the firing of those origins. Furthermore, origin efficiency estimates from this technique correlate well with estimates derived from microarray studies and a recent study in budding yeast showed that checkpoint activation does not disrupt the overall pattern of origin activation (Heichinger et al., 2006 blue right-pointing triangle; Alvino et al., 2007 blue right-pointing triangle). Therefore, we do not believe that HU treatment affects the efficiency of the early firing events that we are studying. We attempted to confirm our results by measuring origin efficiency using two-dimensional (2D) gels, but we found that 2D gels are not sufficiently quantitative to accurately measure the changes in efficiency that we see by combing.

Our results suggest that Hsk1-Dfp1 kinase activity, and presumably the phosphorylation of pre-RCs by Hsk1-Dfp1, is rate-determining in origin activation. As such, phosphorylation by Hsk1-Dfp1 is an excellent candidate for the stochastic step that leads to the random distribution of origin firing in fission yeast. Specifically, we propose that Hsk1-Dfp1 is limiting, but freely diffusible in the nucleus, and thus that it randomly interacts with origins during S phase; when these interactions lead to productive phosphorylation, the origin fires. This model accounts for the stochastic activation of origins and for the effects of increasing and decreasing Hsk1-Dfp1 activity on the probability of activation. It is possible that an earlier stochastic event determines the order of origin firing and that Hsk1-Dfp1 simply executes that order, but such a model seems unnecessarily complicated. Our results are consistent with recent work showing that Cdc7-Dbf4, the budding yeast DDK, is required for efficient origin firing (Hoang et al., 2007 blue right-pointing triangle).

It should be noted that our observation that Hsk1-Dfp1 is freely diffusible in the nucleus does not contradict the observation that Hsk1-Dfp1 binds to chromatin (Takeda et al., 1999 blue right-pointing triangle). All chromatin-binding proteins have a finite dissociation constant; their mobility in the nucleus is determined by the equilibrium between bound and unbound states. That the diffusion coefficient for Dfp1-GFP is approximately threefold lower than GFP alone suggests that Dfp1 spends ~70% of the time bound to fixed structures in the nucleus, presumably pre-RCs. Nevertheless, the time between chromatin binding allows Dfp1-GFP to rapidly sample the nucleus, diffusing across the nucleus in ~100 ms (Figure 3).

Phosphorylation of the MCM complex by Hsk1-Dfp1 is thought to be a critical trigger in origin activation (Sclafani and Holzen, 2007 blue right-pointing triangle). Therefore, is seems likely the Hsk1-Dfp1 executes its rate-limiting function by directly phosphorylating pre-RCs. To test whether the Hsk1-Dfp1 role in regulating origin efficiency does in fact act locally at origins, we tethered Dfp1 via a Gal4 DNA binding fusion to a specific locus in the genome. We find that tethering of Hsk1-Dfp1 does increase the efficiency of local origins. We saw significant increase in efficiency for origins between 3 and 17 kb away from the tethered kinase (Figure 2, AT3002.8, AT3003, AT2102, and AT2103), but no significant effect on origins 32 kb or further away. In this type of experiment, with one protein (Hsk1-Dfp1) being tethered to DNA to increase its local concentration relative to another protein (an origin bound pre-RC) bound to the same region of DNA, the maximum increase in relative concentration is achieved if the two bound proteins are separated by ~3 or 4 times the persistence length of the chromatin—the persistence length being the length over which the chromatin is too stiff to bend easily (Rippe, 2001 blue right-pointing triangle). The persistence length of yeast chromatin is ~3 kb, so the maximum concentration effect should be ~10 kb from the tethered Gal4-Dfp1 (Rippe, 2001 blue right-pointing triangle; Dekker et al., 2002 blue right-pointing triangle). The relative concentration effect falls off approximately exponentially with distance; a site 17 kb away from the tethered Hsk1-Dfp1 (such as AT2102) should have about one third the relative concentration of a site 10 kb away, whereas a site 32 kb away (such as AT2104) should have about 1/30th the concentration (Rippe, 2001 blue right-pointing triangle). These calculations make it plausible that AT2102 would show a strong effect of Hsk1-Dfp1 tethering 17 kb away, but AT2104 would not show an effect of Hsk1-Dfp1 tethering 32 kb away. Therefore, we conclude that Hsk1-Dfp1 acts locally to regulate origin efficiency, presumably by phosphorylating MCM. However, Hsk1-Dfp1 could affect origin efficiency indirectly by phosphorylating other localized regulatory proteins, such as checkpoint kinases, which have been implicated in origin firing in other systems (Shechter and Gautier, 2005 blue right-pointing triangle).

It is interesting to note that, although global and local increase of Hsk1-Dfp1 concentration increases origin efficiency, it does not increase efficiency to close to 100%. This outcome could result from not expressing enough Dfp1 to activate all licensed pre-RCs. Alternatively, it is possible that when Hsk1-Dfp1 is in excess some other factor becomes limiting for origin efficiency. For example, if ORC only binds to a specific origin in 60% of cells, that origin will never be >60% efficient. Origin efficiency in fission yeast roughly correlates with AT richness (Segurado et al., 2003 blue right-pointing triangle; Dai et al., 2005 blue right-pointing triangle). Because ORC is thought to bind AT tracts through the AT-hook motifs in Orc4, this result suggests that ORC binding is an important determinant in origin efficiency (Chuang and Kelly, 1999 blue right-pointing triangle; Dai et al., 2005 blue right-pointing triangle). Other limiting factors, such as ORC occupancy, may explain why some origins are more efficient than other (Feng et al., 2006 blue right-pointing triangle; Heichinger et al., 2006 blue right-pointing triangle; Eshaghi et al., 2007 blue right-pointing triangle; Hayashi et al., 2007 blue right-pointing triangle; Mickle et al., 2007 blue right-pointing triangle). If Hsk1-Dfp1 interacts equivalently with most origins, those that are more likely to be licensed will be more likely to fire. However, other origins, such as the cryptic ars727, may be regulated by cis-acting motifs which could regulate Hsk1-Dfp1 accessibility (Yompakdee and Huberman, 2004 blue right-pointing triangle).

Implications for the Regulation of Replication Timing

Our results show that the Hsk1-Dfp1 replication kinase is rate determining for origin firing in fission yeast, and they explain how both the stochastic and inefficient character of origin firing could result from the random activation of licensed origins by Hsk1-Dfp1 phosphorylation. This model also provides a plausible mechanism for differential regulation of origin efficiency within a cell, in which the efficiency of specific origins is regulated by their accessibility to Hsk1-Dfp1. A similar model has been proposed for origin regulation in budding yeast in which DDK “limited in amount and concentrated at origins would act locally to give the pace at which individual origins fire” (Nougarede et al., 2000 blue right-pointing triangle). Regulation of DDK origin phosphorylation could be positive, by increasing the affinity of Hsk1-Dfp1 for certain origins, or negative, by restricting access of Hsk1-Dfp1, for example by sequestering origins within condensed chromatin (Goren et al., 2008 blue right-pointing triangle). Regulating origin efficiency has been proposed as a mechanism for controlling origin timing (Hyrien et al., 2003 blue right-pointing triangle; Rhind, 2006 blue right-pointing triangle; Bechhoefer and Marshall, 2007 blue right-pointing triangle; Lygeros et al., 2008 blue right-pointing triangle). Therefore, the regulation of origin efficiency via the accessibility of DDK may provide a mechanistic link between chromosome structure and replication timing. Origins in transcriptionally active chromatin, which tend to replicate early in S phase, would fire earlier because they are more accessible to DDK, whereas origins in heterochromatic regions would be less accessible to DDK and thus be less efficient and later replicating. It should be noted that other diffusible, rate-limiting activators could be regulate origin firing in other organisms with the same general consequences for efficiency and timing. For example, both CDK activity and Cdc45 have been suggested to be limiting for origin firing in frog embryo extracts (Krasinska et al., 2008 blue right-pointing triangle).

Supplementary Material

[Supplemental Materials]

ACKNOWLEDGMENTS

We thank members of the Rhind laboratory, in particular, Chaitali Dutta and Mary Porter-Goff, for useful suggestions and experimental help, Jeff Nickerson and Krishnan Kota for help with the photobleaching experiments, Hisao Masai for the Dfp1 antibody, Susan Forsburg for hsk1–1312, John Woodward at the Sanger Center for cosmids, Job Dekker for helpful discussions about the effects of tethering on local concentration, and Sean Ryder and Bill Kobertz for critical reading of the manuscript. This work was funded by National Institutes of Health (NIH) grant GM-069957 (to N. R.) and NIH grant GM-076272 (to J. L.), as well as National Center for Research Resources P40RR01632004 (to J. L.) for funding for a Schizosaccharomyces pombe microarray facility, and a Human Frontier Science Program grant to J. B. and A. B.

Abbreviations used:

BrdU
5-bromo-2′-deoxyuridine
DDK
Dbf4-dependent kinase
HU
hydroxyurea
ORC
origin recognition complex
pre-RC
pre-replication complex
YES
yeast extract plus supplements.

Footnotes

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08-06-0645) on September 17, 2008.

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