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, 
, and 
localize and function together at replication forks in Saccharomyces cerevisiae
,a,bLaboratories for Biomolecular Networks, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresSupplementary materialReferences |
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, -
, and -
. This study examines cell cycle-dependent localization of pol , - and - in Saccharomyces cerevisiae using immuno-histochemical and chromatin immuno-precipitation methods. The results show that pol , -, or - localizes on chromatin as punctate foci at all stages of the cell cycle. However, some foci overlap with or are adjacent to foci pulse-labeled with bromodeoxyuridine during S phase, indicating these are replicating foci. DNA microarray analysis localized pol , -, and - to early firing ARSs on yeast chromosome III and VI at the beginning of S phase. These data collectively suggest that bidirectional replication occurs at specific foci in yeast chromosomes and that pol , -, and - localize and function together at multiple replication forks during S phase. |
Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialReferences |
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, , and (pol , -, and -) (Aparicio et al. 1997; Kawasaki & Sugino 2001; Bell & Dutta 2002). Cdc45 is associated with ARS sequences in G1 and S and interacts with the Mcm2-7 protein complex. This association is dependent on Sld3 and Sld2/Dpb11 (Kamimura et al. 1998; Masumoto et al. 2000). Recently, a novel S. cerevisiae complex was identified called GINS. GINS complex consists of Sld5p, Psf1p, Psf2p, and Psf3p and facilitates initiation of DNA synthesis together with Dpb11, Sld3 and Cdc45 (Takayama et al. 2003). Previous studies (Kesti et al. 1999; Dua et al. 1999) showed that pol2-16 is a viable yeast strain with a deletion of the DNA polymerase domain of pol and cells having only the carboxyl-terminal region of pol are proficient in replication, recombination, and repair of DNA damage. Therefore, it was concluded that although pol may play an enzymatic role during DNA replication, the function is nonessential and that the sole essential function of pol is, instead, provided by its non-catalytic carboxyl-terminal domain. However, pol2-16 grows slower than a wild-type strain and has a prolonged S phase (Dua et al. 1999; Kesti et al. 1999; Ohya et al. 2002). In some strain backgrounds, pol2-16 does not grow at 37 °C, suggesting that the catalytic activity of pol is required for normal replicative DNA synthesis (Ohya et al. 2002).
This study investigates assembly and function of S. cerevisiae pol , -, and - at replication forks during the cell cycle using immunohistochemical and chromatin immuno-precipitation (ChIP) methods. The results show that pol , -, or - localizes on chromatin as punctate foci at all stages of the cell cycle. However, some of each polymerase foci overlap with or are adjacent to sites of nascent DNA synthesis labeled with bromodeoxyuridine (BrdU). Furthermore, we show that pol , -, and - are localized to early firing origin regions during early S phase by use of S. cerevisiae chromosomal DNA microarray chips. These data collectively suggest that bidirectional replication occurs at specific foci in yeast chromosomes and that pol , -, and - localize and function together at multiple replication forks during S phase in S. cerevisiae.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialReferences |
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The factors regulating initiation at different eukaryotic origins of replication are not yet completely understood. For example, it is possible that the localization of replicative DNA polymerases is temporally regulated throughout the cell cycle and that this influences where and when DNA replication initiates during the cell cycle. This was tested by measuring the chromatin-binding properties of DNA pol , -, and - throughout the cell cycle. Whole cell extracts were prepared from cells arrested in G1, S or G2/M using -factor, hydroxyurea (HU) or nocodazole, respectively. Extracts were separated into a soluble and a chromatin-bound fraction, which is DNase sensitive, as previously published (Liang & Stillman 1997) and analyzed by SDS-PAGE and Western using antibodies to each DNA polymerase catalytic subunit.
Figure 1 shows that a chromatin-bound fraction of pol , -, or - was not significantly changed during the cell cycle in yeast, unlike Cdc45. This is unusual, because other replication proteins, such as Mcm2-7, Cdc6, Cdt1, and Cdc45, are specifically recruited to chromatin during G1 and S phase (Liang & Stillman 1997; Zou & Stillman 1998; Bell & Dutta 2002), but are not chromatin-bound during the rest of the cell cycle. Furthermore, expression of DNA pol , -, or - is not constant throughout the cell cycle, but peaks at G1/S (reviewed in Sugino 1995), which is consistent with the expectation that the primary function of the replicative polymerases is during S phase. Thus, this chromatin-binding assay is not useful for understanding when or where pol , -, and - function during the cell cycle.
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, -, or -) catalytic subunit polypeptide (Pol1p, Pol3p, or Pol2p) and Orc1p localize on chromatin as punctate foci in -arrested (F), HU-arrested (HU), and nocodazole-arrested (NOC) cells. Interestingly, the number of Orc1p foci is more or less the same as that of either Pol1p, Pol2p, or Pol3p, although the number of the foci of Orc1p and each polymerase per nucleus varies between G1, early S phase and G2/M phase cells (10–20 foci/F, 30–50 foci/HU, and 30–40 foci/NOC, respectively). These data clearly show that the localization of three replicative DNA polymerases is not dynamically changed during the cell cycle.
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Immunohistochemical method (chromosome spreading followed by immunostaining with antibodies) and labeling of nascent DNA were used to localize yeast replication proteins relative to sites of DNA synthesis on chromatin. Nascent DNA was labeled with bromodeoxyuridine (BrdU) in yeast cells expressing Herpes Simplex Virus thymidine kinase (see Experimental procedures) and sites of BrdU incorporation were detected using monoclonal anti-BrdU antibody. BrdU staining appeared in punctate nuclear foci 20, 40 or 60 min after -factor release (Fig. 3A) (see Supplementary Fig. S1 at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC843/GTC843.htm). The amount of nascent DNA synthesis was estimated by counting BrdU-labeled foci in 100 randomly selected nuclei at each time point. These results are summarized in Fig. 3B. The average number of foci/nucleus was 15 in early S phase, 25 in mid-S phase, and 17 in late S phase. No foci were observed in G1 or G2/M (data not shown), indicating that these foci are likely to be due to replicative DNA synthesis during S-phase.
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Each replicative DNA polymerases and nascent DNA co-localize in nuclear foci during S phase
In a previous publication (Ohya et al. 2002), we showed that some of the replicative DNA polymerase foci overlap with the replication foci labeled in vitro, suggesting that the overlapping foci are the replication foci and that the replicative DNA polymerases, pol , , and function at those replication foci. However, it is not necessarily true that in vitro labeled replication foci is representative of in vivo replication foci. Thus, in this study, we wanted to confirm these results by showing that each replication polymerase localizes at the replication foci labeled in vivo. For this purpose, yeast cells expressing 3xHA-tagged pol (Pol2) and 9xMyc-tagged-pol (Pol1), 9xMyc-pol (Pol3), or 9xMyc-Orc1p were used in the following experiments to analyze proteins associated with BrdU-labeled nuclear foci. Cells were pulse-labeled with BrdU for 5 min, harvested, subjected to chromosome spreading, and chromosomes were probed with BrdU- and HA- or Myc-antibodies. The results show that some but not all foci stain with both BrdU and the replicative enzymes, suggesting that some foci are sites of active replication (Fig. 4A). The results in Fig. 4A were quantified and this analysis is summarized in Fig. 4B. At least more than half of the BrdU foci seen in S phase cells overlap with or locate close to the foci of either pol or -, while about 40% of the BrdU foci overlap with those of pol or Orc1p. From these results, we conclude that foci that lack BrdU staining may not be active sites of DNA replication as the number of the foci of each DNA polymerases exceeds the number of the BrdU foci and that active replication foci are only detected transiently with this system (Fig. 3C). Furthermore, these results also indicate that some active sites of replicative DNA synthesis contain Orc1p, pol , -, or - (as well as other proteins (such as PCNA, RPA, and Rfc2p) whose presence was not measured in this study (S. Hiraga and A. Sugino, unpublished observation). Although we could not carry out quadruple or quintuple labeling experiments to show that all three replicative polymerases co-localize with the replication foci due to lack of proper antibodies with a different fluorescence, it is highly likely that the overlapping foci between BrdU and either pol , -, or - also contain the other replicative polymerases. In any case, the results shown in Fig. 4 are consistent with the notion that all three replicative DNA polymerases (pol , -, and -) function at the replication forks (Sugino 1995; Kawasaki & Sugino 2001; Ohya et al. 2002).
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is loaded on to the replication origin after pol and pol
Previous studies used chromatin immunoprecipitation (ChIP) method to demonstrate that replicative DNA polymerases, PCNA and RPA co-localize with the replication origin and remain associated with replication forks during S phase (Kamimura et al. 1998; Aparicio et al. 1997; Ohya et al. 2002). Here, ChIP was also used to define when pol , -, and - become associated with the origin of replication during S phase. It is very difficult to quantify amount of each protein bound on chromatin by ChIP assay due to different antibodies used for immunoprecipitation and their different accessibility to antigen. However, it was able to estimate timing of each replicative polymerase loading on to replication origin region during the cell cycle. As shown in Fig. 5, pol and pol associate with ARS305 (an early firing origin) at almost the same time, but slightly before pol during early S phase. This pattern was observed both in the absence (Fig. 5A,C) and the presence of HU (Fig. 5D,F). Although the timing difference between pol and pol was small, but was significant (Fig. 5C,F) and highly reproducible. It was estimated to be about 10 min at 20 °C under both conditions. Thus, these results may have implications for the precise roles of pol and - in leading- and lagging-strand DNA synthesis, respectively, during DNA replication (see Discussion).
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, , and at DNA replication fork
ChIP is also a powerful method to study genome-wide distribution of specific proteins (Ren et al. 2000; Wyrick et al. 2001; Katou et al. 2003). Therefore, ChIP was also used here to assess the dynamic associations of replicative polymerases with chromatin during DNA replication. Cells were synchronized at G1 with -factor, S with HU, or G2/M with nocodazole, fixed with formaldehyde, harvested, and disrupted by sonication. Cross-linked DNA fragments were immunoprecipitated with antibody to epitope-tagged protein, which generated a chromatin fraction enriched in binding sites for each protein of interest. Cross-links were reversed and the enriched DNA fractions were amplified using random primers in the presence of Cy5. Total genomic (unenriched) DNA extracted from yeast cells was sonicated, amplified and labeled with Cy3. Enriched (Cy5) and total (unenriched) (Cy3) pools of labeled DNA were cohybridized to a single DNA microarray and the Cy5/Cy3 fluorescence intensity ratio was measured as in Experimental procedures. Three independent experiments were performed and a weight average method was used to calculate the relative affinity of the protein of interest to probe sequences on the array.
The DNA microarray was prepared with 1 kb PCR probes giving approximately 85% coverage of yeast chromosomes III (315 kb) and VI (265 kb). The quality of the DNA microarray was examined and the ChIP system characterized by testing whether previously characterized functional ARS sequences could be detected. Yeast cells expressing 9xMyc-tagged Orc1p were cultured and a DNA fraction enriched in Orc1p binding sites was analyzed as discussed above. This analysis detected Orc1p bound to all known functional ARS sequences on chromosomes III and VI (Raghuraman et al. 2001; Yabuki et al. 2002) (see Supplementary Fig. S2 at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC843/GTC843.htm).
ChIP analysis was then carried out using yeast cells expressing 3HA-tagged pol and 9xMyc-tagged pol , or -. The results are shown in Fig. 6. When ChIP analysis was carried out using cells arrested in G1 or G2/M, no specific origin region bindings were detected for chromosome III (Fig. 6A,B) or chromosome VI (data not shown). However, when cells were arrested in early S phase cells with 0.2 M HU for 60 min after release from -factor, specific binding of three replicative DNA polymerases was detected at early firing origins of replication (Fig. 6C). The hybridization signal was higher when antibody to epitope-tagged pol was used for immunoprecipitation than when the antibody was targeted to epitope-tagged pol or pol . This result may reflect different roles for pol , -, and - at active DNA replication forks. Alternatively, these may reflect antibody differences or accessibility of antibody to the antigens on chromatin. However, the latter possibility is less likely, as we used the same amount of chromosomal DNA precipitated by antibodies for labeling and hybridization analysis.
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, -, and - participate in a DNA synthesis reaction at active replication forks. However, it is also known that pol plays a role as an S phase checkpoint protein (Navas et al. 1995), and its presence at the replication fork might reflect that role. Therefore, this possibility was tested by carrying out ChIP analysis in pol2-16 mutant cells. The pol2-16 mutation carries a deletion in the pol DNA polymerase domains (Dua et al. 1999; Kesti et al. 1999), which is temperature sensitive for growth and causes delayed DNA replication even at permissive temperatures, but which does not impair S phase or DNA damage-induced checkpoints (Dua et al. 1999; Kesti et al. 1999; Ohya et al. 2002). As shown in Fig. 7, wild-type pol and the mutant pol2-16p associated with early firing origins on chromosome III at 45 min after release from -factor in the presence of HU. However, pol2-16p was not any more associated with these origins at 60, 75, or 90 min after release from -factor, while wild-type pol stays on early firing origins regions for at least 90 min after -factor release (Fig. 7B). In contrast, pol and - remained associated with early replicating origins on chromosome III for 45–90 min as in wild-type cells (Supplementary Fig. S3). Similar results were obtained for chromosome VI (data not shown). These results are consistent with the view that mutant pol2-16p interacts with replication origins in an aberrant manner in the presence of HU due to its deleting DNA polymerase domains from pol . Therefore, we conclude that the binding of wild-type pol at replication origins is primarily related to its function of DNA synthesis, but not related to its S-phase checkpoint function. Thus, it is likely that pol substitutes for pol (i.e., in the leading strand synthesis) in the mutant cells; however, the amount of pol and - bound to chromatin does not increase significantly in pol2-16 mutant cells (data not shown). In addition, at some early firing-replication origins, pol fluorescence-label extended outward in both directions from the origin after prolonged incubation (45–90 min) in the presence of HU (Fig. 7A). This is consistent with other studies indicating that DNA replication forks migrate approximately 5 kb in both directions from early firing origins in the presence of HU (Bousset & Diffley 1998; Santocanale & Diffley 1998; Yabuki et al. 2002).
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Discussion |
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, -, and - co-localize with replication foci
This study investigates the subcellular localization of yeast replicative pol , -, and - using immunohistochemical method and ChIP assay. One surprising result was that a fraction of pol , -, and - remain chromatin-bound during the cell cycle (Fig. 1). Consistent with this result, we demonstrated that these three DNA polymerases are present in punctate nuclear foci throughout the cell cycle (Fig. 2), which has also been observed for Orc1p (Fig. 3; Kawasaki et al. 2000; Ohya et al. 2002). Some foci stain with antibody to each polymerase and contain nascent DNA (Fig. 4). Significantly, the number of these overlapping foci increases during S phase (Fig. 4). These foci were most prominent in mid-S phase, when more than half of the BrdU-labeled foci stain with antibody to pol or -, but less than half (about 40%) of the BrdU-labeled foci stain with antibody to pol or Orc1p (Fig. 4). The reason why all BrdU foci did not overlap or locate close to those of either DNA polymerases might be due to just finishing DNA replication foci, which do not contain any DNA polymerases. Alternatively, we used a heat-denaturing step to detect BrdU labeled DNA with BrdU-antibody (see Experimental procedures), so that this step may destroy some of the overlapping foci between BrdU and each DNA polymerases or Orc1p. In any case, due to lack of proper antibodies with a different fluorescence, we could not carry out quadruple or quintuple labeling to show that all three replicative polymerases co-localize with the replication foci. Nevertheless, it is highly likely that the overlapping foci between BrdU and either pol , -, or - also contain the other replicative pols, as it was shown that all three replicative DNA polymerases (pol , -, and -) bind at early firing origins in early S phase by ChIP and ChIP on DNA chip assays (Figs 5–7). Thus, these results strongly suggest that these overlapping foci are sites of active chromosomal DNA replication and contain all three replicative DNA polymerases.
DNA polymerases , - and - function at the replication forks
ChIP is a powerful method to measure protein–DNA interactions on a genome-wide scale. Here, ChIP analysis was used to assess the interactions of pol , - and - with ARS sequences on yeast chromosomes III and VI. The results clearly demonstrate that pol , -, and - bind to early firing origins in early S phase (Figs 6C and 7A), but not in other stages of the cell cycle (Fig. 6A,B). The amount of pol and pol localized to DNA replication origins was significantly lower than the amount of pol (Fig. 6C), suggesting possible functional differences for these polymerases at the replication fork. For example, pol and - could have a more dynamic interaction with chromatin because lagging strand DNA synthesis may require multiple cycles of binding, dissociation, and rebinding of the lagging strand DNA synthesis apparatus to chromosomal DNA (Bae & Seo 2000; Ayyagari et al. 2003; Garg et al. 2004). In contrast, pol may catalyze processive DNA synthesis on the leading DNA strand and may undergo only one cycle of binding/dissociation per replicon per cell cycle. Furthermore, if pol is recruited to the replication origin region by concerted action of Cdc45-Sld3, Dpb11-Sld2, and GINS complex during the initiation of chromosomal DNA replication (Kamimura et al. 1998; Masumoto et al. 2000; Takayama et al. 2003) before Pol is recruited to the replication fork with the help of RFC and PCNA at the primer, it may be a determination factor which DNA polymerase (either pol or pol ) can synthesize the leading strand DNA by using a first RNA-DNA small primer, since each strand of chromosomal DNA is synthesized by either pol or -in vivo (Morrison & Sugino 1994; Shcherbakova & Pavlov 1996; Karthikeyan et al. 2000). Alternatively, these differences may be due to efficacy of each antibody action. However, it is less likely as we observed a similar number of either pol or pol foci to those of pol by chromosome spreading methods (Fig. 4). Furthermore, we used the same amount of Cy5-labeled DNA prepared from different antibodies was used for ChIP on DNA chip analysis.
Pol plays important roles in yeast DNA replication and DNA repair, but it is also involved in sensing DNA damage during S phase. For example, mutations in the C terminus of the catalytic subunit of pol block induction of a RNR3 in response to DNA damage or hydroxyurea (Navas et al. 1995). Therefore, we tested whether pol binding to replication origin association is due to its DNA replication checkpoint function rather than its DNA synthesis function using the pol2-16 mutant, which is a viable yeast strain with a deletion of the DNA polymerase domains of pol , is normal for DNA damage dependent checkpoint as well as for DNA replication checkpoint (Dua et al. 1999; Kesti et al. 1999). Previously, we showed that progression of replication forks is severely retarded in pol2-16 cells under permissive conditions by ChIP assay with PCNA and RPA (Ohya et al. 2002). In this study, by ChIP on DNA chip assay, we also showed that mutant pol2-16p binds transiently to early firing replication origins, while wild-type pol stably binds to early firing origins for quite a long time in the presence of HU (Fig. 7A). Although it is still possible that the replication machinery is much more unstable in pol2-16 mutants than that in wild-type cells, these results show that the pol2-16p, which is the truncation protein of pol that does not have DNA polymerase activity as well as its associated 3'-5' exonuclease activity, has a different binding mode to early firing origins from those of pol , supporting the review that pol binding to origin regions is related to the function of DNA synthesis during S phase. Furthermore, these results strongly suggest that elongation of leading strand DNA synthesis is defective in pol2-16 cells, and that another replicative polymerase, probably pol , provides partial complementation for this defect during replicative DNA synthesis. However, the complementing polymerase, pol , may be less efficient in this role than pol due to lack of its specific interaction between GINS (T. Seki and A. Sugino, unpublished observation) and Mcm2-7 complex, which is believed to be a helicase that functions at the replication fork. At higher temperatures that promote faster fork movement, pol may be insufficient to complement the pol2-16 defect; thus the mutant cells are temperature-sensitive for their growth. In contrast, in temperature-sensitive pol2-9 or pol2-18 mutant cells, which have a temperature-sensitive DNA polymerase activity, the mutant DNA polymerase blocks replication fork movement at the restrictive temperatures, which prevents substitution of pol . In any case, we conclude that co-ordinate function of pol with two other replicative DNA polymerases at the replication fork is disrupted in pol2-16 mutant cells, resulting in slow progression of replication forks and the functional (DNA polymerase active) pol is required for normal chromosomal DNA replication in S. cerevisiae, which contradicts an earlier view on this question (Kesti et al. 1999).
It is known that pol is localized to distinct nuclear foci throughout the cell cycle in human cells, unlike PCNA. And, it has been recently reported that early in S phase, pol does not co-localize with PCNA and newly replicated DNA, but is localized in adjacent or neighboring regions. However, late in S-phase, pol does co-localize with PCNA and nascent DNA (Fuss & Linn 2002). These observations suggest that pol and PCNA have separate but associated functions early in S phase and coordinated functions late in S phase (Fuss & Linn 2002). In this paper, we showed that pol as well as pol and pol localizes to early firing origins in early and late-firing origin in late S-phase (Figs 5–7). Thus, it is highly possible that pol participates in replicating all regions of yeast chromosomes, unlike involving in DNA synthesis in specific regions of yeast chromosomes.
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Experimental procedures |
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All strains are in the W303-1 A background (obtained from R. Rothstein) (MATa ura3-1 trp1-1 leu2-3, 112 his3-11, 15 ade2-1 can1-100) carrying HA or c-Myc epitope-tagged DNA polymerase gene. These were SHY35 (POL2-3HA 
bar1), SHY47 (POL2-3HA POL3-9Myc bar1), SHY54 (POL1-9Myc POL2-3HA bar1), SHY41 (POL3-9HA ORC1-3HA bar1), SHY46 (POL1-9Myc ORC1-3HA bar1), SHY47 (POL2-9Myc ORC1-3HA bar1), SHY63 (MATa Gal promoter-TK::TRP1 ORC1-3HA::URA3 POL2-9Myc::LEU2 bar1), SHY67 (MATa Gal promoter-TK::TRP1 POL1-3HA::URA3 POL3-9Myc::LEU2 bar1), and SHY41 (W303-1 A pol2::pol2-16--3HA::URA3 POL3-9Myc::LEU2 bar1).
Yeast media and synchronization
Media and cell synchronization were previously described (Kamimura et al. 1998; Ohya et al. 2002; Takayama et al. 2003). Cells were grown to a density of 1.0–1.3 x 107 cells/mL in YPD at 25 °C. For -factor arrest (G1 arrest), cells were suspended in YPD with 30 ng/mL -factor, incubated for 3 h at 25 °C. For G2/M arrest, nocodazole was added to final concentration of 10 µg/mL and cells were incubated for 2 h at 25 °C. Hydroxyurea (HU)-treated cells were arrested by -factor as indicated above, and HU was added to final concentration of 0.2 M and incubation was continued for 1 h at 25 °C. Cells were washed, re-suspended in YEPR (Ohya et al. 2002) containing 0.2 M HU and 0.1 µg/mL of Actinase E and incubated for 1.5–2 h at 25 °C.
Bromodeoxyuridine (BrdU) labeling
Yeast SHY63 or SHY67 cells were grown to 1 x 106 cells/mL in synthetic-galactose complete medium at 25 °C without leucine, tryptophan or uracil. Cells were synchronized with 30 ng/mL -factor for 3 h, released into medium containing 10 µg/mL Actinase E. At indicated time points, cells were labeled with BrdU for 5 min and chased by dilution in 9 volumes medium containing 1 µg/mL thymidine and incubation for 2.5 min. Sodium azide was added to 0.1% and cells were collected by centrifugation. Cells were spheroplasted and chromosome spreads were prepared on glass slides as described (Kawasaki et al. 2000; Ohya et al. 2002). Slides were dried, heated for 2.5 min at 98 °C and quickly chilled in distilled water. Immunostaining procedures were previously described (Kawasaki et al. 2000; Ohya et al. 2002).
Focus scoring and statistical analyses
Color composite images from two-dye experiments were produced and scored for co-localization of dye-specific signals as previously described (Gasior et al. 1998; Kawasaki et al. 2000; Ohya et al. 2002).
ChIP (chromatin immuno-precipitation) assay
ChIP assay was carried out as described (Tanaka et al. 1997) with some modifications (Kamimura et al. 1998). Yeast cells (5–8 x 108) were fixed in 1% formaldehyde for 20 min at room temperature and washed twice with ice-cold TB buffer [20 mM Tris-HCl (pH 7.6)/0.2 M NaCl]. Cells were suspended in 1 M sorbitol/0.1 M EDTA/20 mM DTT and lyzed with 130 µg/mL Zymolyase 100T (Seikagaku, Japan) for 5 min at 30 °C. Cells were washed once with sorbitol solution and re-suspended in lysis buffer (20 mM HEPES-KOH (pH 7.5)/140 mM NaCl/1 mM EDTA/1% Triton X-100/0.5% sodium deoxycholate/1 x Complete Protease Inhibitors Cocktail (Roche)/1 mM PMSF). Cells were sonicated with an ultrasonic disrupter (Tomy, Japan) at maximum power with 10 pulses x 4 on ice, yielding 300–900 bp chromatin fragments. The solution was clarified by centrifugation and the supernatant fraction was used as the whole cell extract (WCE). WCE was divided into two aliquots; one aliquot portion was incubated at 4 °C overnight with 10 µL Protein A magnetic beads (Dynal) and 2 µg anti-HA monoclonal antibody (12CA5). The second aliquot of WCE was incubated with 10 µL of Protein A magnetic beads (Dynal) and 2 µg of anti-Myc monoclonal antibody (9E11). Beads were washed sequentially with lysis buffer, lysis buffer containing 210 mM NaCl and washing buffer (10 mM Tris-HCl (pH 8.0)/250 mM LiCl/1 mM EDTA/0.5% NP-40/0.5% sodium deoxycholate/1 x complete protease inhibitors cocktail/1 mM PMSF). DNA was recovered from beads by treating with 125 µg/mL Proteinase K at 65 °C for 2 h and extracted twice with phenol/chloroform. DNA was ethanol-precipitated and resuspended in 0.1 mL TE/1 µg/mL RNase A.
Microarray preparation
Microarray probes for S. cerevisiae chromosomes III and VI were generated using 1172 PCR primers (each primer sequence is available upon request) for 528 PCR products, each one kb in length, based on the Saccharomyces Genome Database (http://genome-www2.stanford.edu/cgi-bin/SGD/web-primer). Size and concentration of PCR products were determined by agarose gel electrophoresis. Probes were spotted on to poly L-lysine-coated glass slides (TaKaRa, Japan) using an Affymetrix 417 Arrayer (Affymetrix). Control probes were for KAR1. The microarray achieved 89% coverage of chromosomes III and VI.
Probes for microarray
Microarray probes were synthesized as described by P. Brown (http://cmgm.stanford.edu/pbrown/protocols/) with modifications. DNA was purified from chromatin immunoprecipitates of yeast G1 arrested, HU-treated, or G2/M arrested cells. ChIP assays were carried out with DNA polymerase-specific antibodies annealed with 100 pmol A1 primer (5'-GGAATTCCAGCTGACCACCNNNNNNNNN-3') and annealed primers were elongated twice by T7 DNA polymerase (Sequenase Version 2.0, USB) at 37 °C for 8 min. The products were amplified again with 100 pmol A2 primer (5'-GGAATTCCAGCTGACCACC-3') for 32 cycles (40 °C, 30 s; 50 °C, 30 s; and 70 °C, 1 min). PCR products were 300–1500 bp in size. The amplified DNA was labeled with Cy3-or Cy5-conjugated dCTP using Klenow fragment of E. coli DNA PolI using the BIOPRIME DNA labeling System (Invitrogen). Cy3- or Cy5-labeled DNA was hybridized for 16 h at 65 °C to yeast chromosome III/VI microarray. The microarray was washed at 65 °C with 2 x SSC/0.02% SDS for 5 min, 1 x SSC for 5 min, and 0.2 x SSC for 5 min. The plate was dried by centrifugation at 1000 r.p.m. for 1 min.
Scanning and analysis of microarray
Microarrays were scanned using an Affymetrix 418 scanner. Hybridization signals were normalized and processed by ImageneTM software (BioDiscovery Inc., CA, USA).
Antibodies
Antibodies used in this study were: monoclonal mouse anti-BrdU (clone B44) (Becton Dickinson), monoclonal mouse anti-HA antibody 16B12 (Berkeley Antibody Company), polyclonal rabbit anti-HA (Santa Cruz Biotechnology), polyclonal rabbit anti-Myc (Cell Signaling Technology), and polyclonal rabbit anti-Myc antiserum (Medical & Biological Laboratories, Co., Japan). Fluorescence-conjugated second antibodies were Alexa488-conjugated goat anti-rabbit IgG (Molecular Probes), and Alexa546-conjugated goat anti-mouse IgG (Molecular Probes).
Other experimental procedures
BrdU was purchased from Sigma-Aldrich, Co. Other Experimental procedures used in this study were previously described (Kamimura et al. 1998; Ohya et al. 2002; Takayama et al. 2003).
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Supplementary material |
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http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC843/GTC843sm.htm.
Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
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Acknowledgements |
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Footnotes |
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aPresent address: Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK.
bPresent address: Communications Research Laboratory, Kansai Advanced Research Center, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe, Hyogo 651–2492, Japan.
cPresent address: Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
These authors equally contributed to this work.
* Correspondence: E-mail: asugino{at}fbs.osaka-u.ac.jp |
References |
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Ayyagari, R., Gomes, X.V., Gordernin, D.A. & Burgers, P.M. (2003) Okazaki fragment-maturation in yeast. I. Distribution of functions between FEN1 and Dna2. J. Biol. Chem.
278, 1618–1625.
Bae, S.-H. & Seo, Y.-S. (2000) Characterization of the enzymatic properties of the yeast Dna2 helicase/endonuclease suggests a new model for Okazaki fragment processing. J. Biol. Chem.
275, 38022–38031.
Bell, S.P. (1995) Eukaryotic replicators and associated protein complex. Curr. Opin. Genet. Dev. 5, 162–167.[CrossRef][Medline]
Bell, S.P. & Dutta, A. (2002) DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374.[CrossRef][Medline]
Bousset, K. & Diffley, J.F.X. (1998) The Cdc7 protein kinase is required for origin firing during S phase. Genes Dev.
12, 480–490.
Donovan, S., Harwood, J., Drury, L.S. & Diffley, J.F.X. (1997) Cdc6-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc. Natl. Acad. Sci. USA
94, 5611–5616.
Dua, R., Levy, D.L. & Campbell, J.L. (1999) Analysis of the essential functions of the C-terminal protein/protein interaction domain of Saccharomyces cerevisiae pol and its unexpected ability to support growth in the absence of the DNA polymerase domain. J. Biol. Chem.
274, 22283–22288.
Fuss, J. & Linn, S. (2002) Human DNA polymerase colocalizes with Proliferating Cell Nuclear Antigen and DNA replication late, but not early, in S phase. J. Biol. Chem.
277, 8658–8666.
Garg, P., Stith, C.M., Sabonri, N., Johansson, E. & Burgers, P.M. (2004) Idling by DNA polymerase d maintains a ligatable nick during lagging-strand DNA replication. Genes Dev.
18, 2764–2773.
Gasior, S.L., Wong, A.K., Kora, Y., Shinohara, A. & Bishop, D.K. (1998) Rad52 associates with RPA and functions with rad55 and rad57 to assemble meiotic recombination complexes. Genes Dev.
12, 2208–2221.
Kamimura, Y., Masumoto, H., Sugino, A. & Araki, H. (1998) Sld2, which interacts with Dpb11 in Saccharomyces cerevisiae, is required for chromosomal DNA replication. Mol. Cell. Biol.
18, 6102–6109.
Karthikeyan, R., Vonarx, E.J., Straffon, A.F., et al. (2000) Evidence from mutational specificity studies that yeast DNA polymerases and replicate different DNA strands at an intracellular replication fork. J. Mol. Biol.
299, 405–419.[CrossRef][Medline]
Katou, Y., Kanoh, Y., Bando, M., et al. (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424, 1078–1083.[CrossRef][Medline]
Kawasaki, Y. & Sugino, A. (2001) Yeast replicative DNA polymerases and their role at the replication fork. Mol. Cells 12, 277–285.[Medline]
Kawasaki, Y., Hiraga, S. & Sugino, A. (2000) Interaction between Mcm10 and other replication factors are required for proper initiation and elongation of chromosomal DNA replication in Saccharomyces cerevisiae. Genes Cells 5, 975–989.[Abstract]
Kesti, T., Flick, K., Keranen, S., Syvaoja, J.E. & Wittenberg, C. (1999) DNA polymerase catalytic domains are dispensable for DNA replication, DNA repair, and cell viability. Mol. Cell
3, 679–685.[CrossRef][Medline]
Liang, C. & Stillman, B. (1997) Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants. Genes Dev.
11, 3375–3386.
Masumoto, H., Sugino, A. & Araki, H. (2000) Dpb11 controls the association between DNA polymerases and and the autonomously replicating sequence region of budding yeast. Mol. Cell. Biol.
20, 2809–2817.
Morrison, A. & Sugino, A. (1994) The 3'–5' exonucleases of both DNA polymerases and participate in correcting errors of DNA replication in Saccharomyces cerevisiae. Mol. Gen. Genet.
242, 289–296.[CrossRef][Medline]
Navas, T.V., Zhou, Z. & Elledge, S.J. (1995) DNA polymerase links the DNA replication machinery to the S phase checkpoint. Cell
80, 29–39.[CrossRef][Medline]
Ohya, T., Kawasaki, Y., Hiraga, S., et al. (2002) The DNA polymerase domain of pol is required for rapid, efficient, and highly accurate chromosomal DNA replication, telomere length maintenance, and normal cell senescence in Saccharomyces cerevisiae. J. Biol. Chem.
277, 28099–280108.
Raghuraman, M.K., Winzeler, E.A., Collingwood, D., et al. (2001) Replication dynamics of the yeast genome. Science
294, 115–121.
Ren, B., Robert, F., Wyrick, J.J., et al. (2000) Genome-wide location and function of DNA binding proteins. Science
290, 2306–2309.
Santocanale, C. & Diffley, J.F.X. (1998) A Mec1- and Rad53-dependent checkpoint controls late firing origins of DNA replication. Nature 395, 615–618.[CrossRef][Medline]
Shcherbakova, P.V. & Pavlov, Y.I. (1996) 3'–5' exonucleases of DNA polymerases and correct base analog induced DNA replication errors on opposite DNA strands in Saccharomyces cerevisiae. Genetics
142, 717–726.[Abstract]
Sugino, A. (1995) Yeast DNA polymerases and their role at the replication fork. Trends Biochem. Sci. 20, 319–323.[CrossRef][Medline]
Takayama, Y., Kamimura, Y., Okawa, M., Muramatsu, S., Sugino, A. & Araki, H. (2003) GINS, a novel multiprotein complex required for chromosomal DNA replication in budding yeast. Genes Dev.
17, 1153–1165.
Tanaka, T., Knapp, D. & Nasmyth, K. (1997) Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs. Cell 90, 649–660.[CrossRef][Medline]
Wyrick, J.J., Aparicio, J.G., Chen, T., et al. (2001) Genome-wide distribution of ORC and MCM proteins in S. cerevisiae: High-resolution mapping of replication origins. Science
294, 2357–2360.
Yabuki, N., Terashima, H. & Kitada, K. (2002) Mapping of early firing origins on a replication profile of budding yeast. Genes Cells 7, 781–789.[Abstract]
Zou, L. & Stillman, B. (1998) Formation of a preinitiation complex by S-phase cyclin CDK-dependent loading of Cdc45p onto chromatin. Science
280, 593–596.
Received: 27 October 2004
Accepted: 21 December 2004
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