Nucleic Acids Research, 2000, Vol. 28, No. 20 3846-3852
© 2000 Oxford University Press
Structure and function of the fourth subunit (Dpb4p) of DNA polymerase 
in Saccharomyces cerevisiae
Department of Biochemistry and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3–1 Yamada-oka, Suita, Osaka 565-0871, Japan
Received July 17, 2000; Revised and Accepted September 1, 2000.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
DNA polymerase
(Pol) of Saccharomyces cerevisiae is purified as a complex of four polypeptides with molecular masses of >250, 80, 34 (and 31) and 29 kDa as determined by SDS–PAGE. The genes POL2, DPB2 and DPB3, encoding the catalytic Pol2p, the second (Dpb2p) and the third largest subunits (Dpb3p) of the complex, respectively, were previously cloned and characterised. This paper reports the partial amino acid sequence of the fourth subunit (Dpb4p) of Pol. This protein sequence matches parts of the predicted amino acid sequence from the YDR121w open reading frame on S.cerevisiae chromosome IV. Thus, YDR121w was renamed DPB4. A deletion mutant of DPB4 (
dpb4) is not lethal, but chromosomal DNA replication is slightly disturbed in this mutant. A double mutant haploid strain carrying the dpb4 deletion and either pol2-11 or dpb11-1 is lethal at all temperatures tested. Furthermore, the restrictive temperature of double mutants carrying dpb4 and dpb2-1, rad53-1 or rad53-21 is lower than in the corresponding single mutants. These results strongly suggest that Dpb4p plays an important role in maintaining the complex structure of Pol in S.cerevisiae, even if it is not essential for cell growth. Structural homologues of DPB4 are present in other eukaryotic genomes, suggesting that the complex structure of S.cerevisiae Pol is conserved in eukaryotes. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In Saccharomyces cerevisiae, three DNA polymerases,
, 
and , participate in chromosomal DNA replication (see 1 for a review). DNA polymerase (Pol) consists of four subunits [Pol1p (Cdc17p), Pol12p, Pri1p and Pri2p] and its primary role is in the initiation of DNA replication and priming of Okazaki fragments. DNA polymerases and (Pol and Pol) are required to complete synthesis of both the leading and lagging strands. Consistent with this notion, it has been shown by chromatin immunoprecipitation assay (2,3) that both Pol and Pol bind at/or near replication origins and move with the replication fork. However, the precise functions performed by Pol and Pol on leading and lagging strands have not yet been defined (1,4,5).
Saccharomyces cerevisiae Pol is a multi-subunit complex that contains the three subunits Pol3 (Cdc2) (5), Hys2 (Pol31) (6) and Pol32 (6,7), which are homologues of Schizosaccharomyces pombe Pol3, Cdc1 and Cdc27, respectively (8). Pol requires additional replication factors, PCNA and RF–C complex, to catalyse processive DNA synthesis, suggesting that Pol is the leading strand polymerase (see 5 for a review). Pol also has a 3'
5' exonuclease activity that acts as an editing function during DNA synthesis (9,10).
Pol is also a multi-subunit complex consisting of Pol2p, Dpb2p, Dpb3p and Dpb4p (1,11,12). Although Pol itself is a highly processive enzyme, it also requires PCNA and RF–C complex to catalyse processive DNA synthesis under certain conditions (11,13,14). The Pol2p protein is the catalytic subunit and is encoded by the POL2 gene (15). It is a class B polymerase, characterised by a series of conserved domains, called domains I–VI, containing the 3'5' exonuclease subdomains and the DNA polymerase active site residues in the N-terminal half of the protein (16). Yeast strains with the pol2-9 or pol2-18 mutations within the polymerase domain in POL2 are temperature-sensitive for chromosomal DNA replication (17).
POL2 also includes a large region that is conserved in Pol from all organisms but is not found in any other class B polymerase. The C-terminus of POL2 has dual roles in DNA replication and the response to DNA damage during S phase (18), as demonstrated by the fact that the C-terminal pol2-11 and pol2-12 mutants are deficient in both processes (18–20). In addition, suppressor studies, synthetic lethal tests and purification of various Pol subassemblies from yeast (21–23) have shown that the C-terminal half of POL2 is important in the assembly of the Pol holoenzyme [PolII* (11)].
The second subunit of Pol (Dpb2), encoded by the DPB2 gene, is essential for yeast cell growth and required for chromosomal DNA replication (12). The DPB3 gene encodes the third subunit of Pol; this subunit is not essential for yeast cell growth, although it plays an important role in stabilising the subunit structure of the polymerase (21).
It was recently shown that Pol along with its interacting proteins, Dpb11p and Sld2p, plays an important role bringing other polymerase complexes and replication proteins to replication origins before elongation of DNA strands (3,23). More recent studies have shown that although Pol may play an enzymatic role during DNA replication, this function does not appear essential, whereas the essential function of Pol is provided by its non-catalytic C-terminal domain (24,25). Nonetheless, Pol, like Pol, has the 3'5' exonuclease activity that provides editing function during DNA synthesis. Both Pol and Pol 3'5' exonuclease-deficient yeast strains are spontaneous mutators (see 16 for a review), strongly suggesting that both polymerases are involved in normal chromosomal DNA replication. The subunit structure of Pol in other eukaryotic systems including the fission yeast S.pombe (26) is not well characterised yet, although it is known that calf thymus Pol also includes at least the second subunit (27).
In this paper, the gene encoding the fourth subunit of Pol is identified and characterised for the first time. The fourth subunit (Dpb4p) of Pol is encoded by the YDR121w open reading frame on the S.cerevisiae chromosome IV. Although the gene is not essential for cell growth, its gene product plays an important function not only in maintaining the complex structure of Pol, but also in interactions between Pol and other replication proteins in the cell.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Yeast strains
Yeast S.cerevisiae strains used in this study are shown in Table 1. Yeast cells were grown in YPD medium (28) unless otherwise noted. In order to maintain selectable plasmids, yeast cells were grown in SD complete medium (28) minus the selectable amino acid(s) or uracil.
|
Expression of Dpb4 protein in Escherichia coli
The NcoI–BamHI DNA fragment containing the YDR121w gene was inserted into pET15b plasmid (Novagen), resulting in the pET15b-DPB4 plasmid. Escherichia coli BL21 (DE3) cells transformed with pET15b-DPB4 plasmid DNA were grown at 37°C in LB + 0.1 mg/ml ampicillin (900 ml) to 8 x 108 cells/ml and 0.5 mM IPTG was added into the cell culture to induce protein overexpression. After 3 h incubation, cells were collected by centrifugation, suspended in 10 ml buffer A (lacking PMSF) and sonicated at 0°C. The cell lysate was centrifuged at 8000 r.p.m. for 20 min in a Beckman JA20 rotor. The supernatant in the sample buffer was heated at 95°C for 3 min and subjected to SDS–PAGE. The 29-kDa polypeptide was eluted from the gel and identified as Dpb4 protein by protein sequencing. The protein eluted from the gel was used to immunise rabbits as reported previously (6).
Purification of Pol holoenzyme
More than 90% pure Pol holoenzyme [PolII* (11)] (~500 µg) was purified from 2 kg of yeast CB001 cells as described previously (11,14).
Protein sequencing
Pol holoenzyme (200 µg) was fractionated by electrophoresis in a 5% SDS–polyacrylamide gel. After staining the gel with Coomassie brilliant blue, the 29-kDa band (Dpb4p) was excised and electroeluted. The eluted polypeptide (
20 µg) was digested in 0.2 M Tris–HCl (pH 9.5) containing 5 pmol of lysylendopeptidase (Wako) at 37°C overnight. To terminate the proteolysis, 1/10 vol of 10% trifluoroacetic acid was added and the supernatant was collected. Oligopeptides were separated by reverse-phase high-pressure liquid chromatography and were subjected to amino acid sequencing using a PSQ-10 protein sequencer (Shimadzu) as described (6).
Construction of dpb4 mutant
The 5' non-coding region (500 bp) of DPB4 was amplified by PCR using primers containing EcoRI and BamHI sites. Similarly, the 3' non-coding region was amplified by PCR using two primers containing BamHI and HindIII sites, respectively. The amplified DNA fragments were treated with BamHI and ligated together by DNA ligase. The resultant DNA fragments were treated with EcoRI and HindIII and inserted into pUC119 pretreated with the same enzymes. The recombinant plasmid DNA was cut with BamHI, mixed with URA3 excised from YDp-U (29) by BamHI, and ligated together to generate pUC119-dpb4::URA3. Similarly, pUC119-dpb4::TRP1 was constructed using YDp-W (29). Either dpb4::URA3 or dpb4::TRP1 was cut out and used to transform yeast and to replace chromosomal DPB4 with dpb4::URA3 or dpb4::TRP1.
Synchronisation of yeast cells
Either W303-1A bar1 or its derivatives were grown in YPD at 25°C to 1 x 107 cells/ml, transferred to YPD containing 30 ng/ml factor and incubated for 2 h. Cells were harvested by centrifugation and resuspended in YPD containing 0.1 mg/ml Actinase E (Kaken Seiyaku) as described (23). After -factor release, cells were incubated at either 25 or 37°C.
Fluorescence activated cell sorting (FACS) analysis
FACS analysis was performed as described previously (30) after yeast cells were fixed with ethanol and stained with propidium iodide.
Antibodies
Rabbit antiserum against yeast Pol complex was as described previously (21). Rabbit antiserum against Dpb4p expressed and purified from E.coli was raised as described previously (6).
Other methods
Western blotting, SDS–PAGE and other methods used in this study were as previously described (6,14,23).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Identification of the gene encoding the 29-kDa polypeptide (Dpb4) co-purified with Pol
activityAmino acid sequences of two oligopeptides (LDEYQAAVEQR and EHDEIEEQGDALQDVEESSE, where an amino acid is indicated by a single letter) were obtained from the 29-kDa polypeptide (the fourth subunit of Pol
) co-purified with Pol activity. These peptide sequences were used to search Stanford Saccharomyces Genome databases (http://genome-www.stanford.edu/Saccharomyces/ ). A completely identical sequence was found in the polypeptide predicted from the open reading frame YDR121w. The sequence match is located at residues 115–126 and 157–177 of the predicted polypeptide from YDR121w.
In order to prove that YDR121w encodes the 29-kDa polypeptide (Dpb4), a yeast genomic DNA fragment containing YDR121w was cloned and expressed in E.coli as described in Materials and Methods. The expressed polypeptide was used as an antigen to raise rabbit antiserum. As shown in Figure 1, this antiserum specifically recognised the 29-kDa polypeptide that co-purified with Pol2p (>200 kDa polypeptide, the catalytic subunit of Pol) in both partially and highly purified Pol fractions. As described below, a deletion mutant (dpb4) of DPB4 did not express this 29-kDa polypeptide (Fig. 1). These results indicate that the open reading frame YDR121w encodes Dpb4p. We therefore rename it DPB4.
|
DPB4 is not essential for yeast cell growth
To investigate whether DPB4 is essential for cell growth, the open reading frame of the chromosomal gene was replaced with either URA3 or TRP1 by the one step replacement technique. Haploid strains with the deleted DPB4 allele were viable and readily obtained (data not shown), indicating that the DPB4 gene is not essential for cell growth in yeast. The obtained
dpb4 haploid strains were tested for their sensitivity to UV light, X-rays, methylmethane sulfonate and hydroxyurea. The results showed that they were not sensitive to any of those treatments (data not shown). However, a slight growth defect was observed in a dpb4 haploid strain at both 25 and 37°C. The generation times of a dpb4 haploid strain at 25 and 37°C in YPD medium were 210 and 250 min, respectively, while those of a parental wild-type strain at both temperatures were 180 and 140 min, respectively. Logarithmically growing dpb4 mutant and wild-type cells were synchronised by factor (23) and released into fresh YPD medium at either 25 or 37°C. After incubation at either 25 or 37°C, cells were harvested, fixed with ethanol, stained with propidium iodide and analysed by a FACScan. As shown in Figure 2, a slightly prolonged S phase was observed in dpb4 mutant cells at 25°C. This prolonged cell cycle progression in S phase was enhanced at 37°C. A majority of dpb4 mutant cells were still in G1/S phase 90 min after factor release at 37°C, when wild-type cells were already in G2/M phase (Fig. 2). These results suggest that although the DPB4 gene is not essential for yeast cell growth, its gene product plays an important role in chromosomal DNA replication.
|
Pol
complex is less stable in dpb4 mutant cells than in wild-type cellsTo prove that Dpb4p is missing in
dpb4 mutant cells, Pol activity was partially purified from dpb4 mutant cells by MonoQ column chromatography as described (11). As shown in Figure 1A, Pol activity was detected in fractions different from those of wild-type Pol. Furthermore, the total amount of Pol from dpb4 mutant cells was ~50% of Pol from wild-type cells. The fractions containing the activity were subjected to western blot, and probed with Pol complex and Dpb4p antibodies. As shown in Figure 1B and C, no Dpb4p was detected in the fractions where a degraded form of Pol and Pol activities eluted. Furthermore, an intact form of Pol (which contains >250 kDa Pol2 polypeptide) could not be separated from a degraded form of Pol (~145 kDa polypeptide) by MonoQ column chromatography, which does not occur in wild-type cells (11,14). In addition, both Dpb2p and Dpb3p dissociate more easily from the Pol2 polypeptide after MonoQ chromatography in extracts from dpb4 mutant cells, resulting in a much smaller amount of Dpb3p rather than wild-type Dpb3p being associated with Pol2p polypeptide (Fig. 1B). Therefore, these results suggest that Pol complex is much less stable in dpb4 mutant cells than in wild-type cells. This observation is consistent with the notion that Dpb4 plays an important role in maintaining the Pol complex in the cell.
Biochemical properties of Pol from dpb4 mutant cells
To investigate whether Dpb4p plays a role in an in vitro DNA synthesis catalysed by Pol, we compared biochemical properties of Pol from dpb4 mutant cells with those from wild-type cells using various templates/primers including an activated calf thymus DNA, oligo(dT)10/poly(dA)>500 (1:50) and singly-primed 
X174 viral single-stranded DNA as previously published (11,14). Since we could not separate a degraded form of Pol from Pol complex in mutant cell extracts, we had to use the mixture of the two forms. However, the mixture exhibited the same optimal pH, optimal MgCl2 concentration, salt sensitivity, Km for dNTPs and processivity as those of wild-type Pol complex (data not shown). Therefore, we concluded that Dpb4p does not have any positive role for in vitro DNA synthesis catalysed by Pol.
Genetic interactions between the dpb4 mutant and other DNA replication mutants
As shown in Figure 2, the dpb4 mutant exhibits a slight delay in S phase progression, suggesting that it has a slight defect in chromosomal DNA replication. This prompted us to investigate genetic interactions between dpb4 and other DNA replication mutants. A dpb4 mutant was genetically crossed with other DNA replication mutants. The resultant diploids were sporulated and the tetrads were micro-dissected, germinated at 25°C for 4 days and each of the spores was analysed genetically. When dpb4 was crossed with a temperature-sensitive pol2-11 mutant, no spores containing both dpb4 and pol2-11 were recovered from more than 40 tetrads (data not shown). On the other hand, a haploid spore containing both mutations could be easily recovered from the tetrads, if a single copy plasmid containing either POL2 or DPB4 was introduced into the strain before crossing (data not shown). Thus, these results indicate that a haploid strain with dpb4 and pol2-11 is not viable (synthetic lethal) at 25°C. Similar synthetic lethality was also observed between dpb4 and pol2-12 and dpb4 and dpb11-1 (Table 2 and data not shown). In contrast, double mutant haploid strains were viable with dpb4 and either rad53-1, rad53-21, dpb2-1, pol3-12, hys2-2 or pol32. The growth of these double mutant strains (and the control single mutant strains) was tested on a YPD plate at various temperatures (31). As shown in Table 2, the double mutants dpb4 rad53-1, dpb4 rad53-21, dpb4 dpb2-1, dpb4 hys2-2 and dpb4 pol32 had significantly lower restrictive temperatures than the corresponding single mutant strains. In particular, dpb4 rad53-1 and dpb4 rad53-21 double mutants exhibited a very poor growth phenotype at 25°C. Significantly, a dpb3 dpb4 deletion mutant had the same restrictive temperature as the single deletion mutant dpb3 or dpb4. Nevertheless, these results indicate a wide variety of genetic interactions between the DPB4 and other DNA replication genes, which suggests that Dpb4p plays an important function as the subunit of Pol during chromosomal DNA replication in S.cerevisiae.
|
Dpb4p contains the histone-fold motif
The predicted amino acid sequence of DPB4 is similar to several other predicted proteins in the databases: DPB4 has 30% identity and 55% similarity to a putative DNA binding protein of S.pombe (DDBJ/EMBL/GenBank accession no. CAB09118); 23% identity and 51% similarity to Caenorhabditis elegans putative DNA binding protein (DDBJ/EMBL/GenBank accession no. AAC77511); 26% identity and 50% similarity to Xenopus and human nuclear transcription factor Y (DDBJ/EMBL/GenBank accession nos AAC82336 and NP-006157, respectively); and 26% identity and 50% similarity to mouse CCAAT-binding transcription factor subunit A (CBF-A) (DDBJ/EMBL/GenBank accession no. P25209). These six proteins were aligned using the program ClustalW 1.7 (32) and the result is shown in Figure 3. The block Maker program identified one conserved region at the N-terminal end of the protein that contains a histone-fold motif (shown by a double-line in the figure). The histone-fold motif resembles an extended helix–strand–helix motif first identified in the core histone proteins as primarily responsible for dimerisation of the H2A/H2B and H3/H4 histone pairs (33). Recently, this motif has been found in a number of proteins involved in protein–protein and protein–DNA interactions (34).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This paper presents evidence that the YDR121w open reading frame on S.cerevisiae chromosome IV encodes the fourth subunit of Pol
complex. Therefore, YDR121w has been renamed DPB4. In addition, it is shown that Dpb4p (Fig. 3) and Dpb3p (our unpublished results) contain the histone-fold motif that is also present in many other proteins. The histone-fold motifs of Dpb4p and Dpb3p are related to those of subunits A and C, respectively, of human CCAAT binding factor CBF. An essential function of the histone-fold motifs of the CBF subunits is to create a protein–protein interaction surface for binding to the histone acetyltransferase enzymes such as GCN5 and p/CAF (35). These histone acetyltransferases can activate CBF transactivation in vivo, possibly by acetylation of the N-terminal lysine residues of histones which disrupts local chromatin structure, thereby facilitating CBF access to CCAAT promoter sites. At this time there is no evidence that Dpb3p and Dpb4p are part of the CCAAT binding factor; however, Dpb3p and Dpb4p may also create a protein–protein interaction surface allowing the Pol complex to interact with other proteins, such as the Dpb11p–Sld2p complex (23), which could also modify chromatin structure. Consistent with this, dpb4 genetically interacts with many other replication mutants (Table 2). Among those genetic interactions, the interaction between dpb4 and dpb11-1 is very strong. This strong interaction suggests that two proteins physically interact inside the cell. In fact, previous works showed that Dpb11p physically interacts with Pol complex during S phase (23). The interaction between dpb4 and pol2 is allelic specific. Only pol2-11 and pol2-12 mutations, but neither pol2-9, pol2-18 or pol2-16, showed synthetic lethality with dpb4 mutation (Table 2 and our unpublished results). Thus, these results strongly suggest that Dpb4p physically interacts with the C-terminal portion of Pol polypeptide. Other weak interactions observed between dpb4 and other DNA replication mutants may reflect their indirect interactions. Interestingly, no physical or genetic interaction has been detected between Dpb3p and Dpb4p (Table 2 and our unpublished results). Therefore, it is possible that Dpb3p and Dpb4p function independently.
Compared to the extensive knowledge of the influence of histone acetylation on transcription, little is known of its influence on chromatin structure in replication. It is possible that local disruption of chromatin structure by histone acetylation might facilitate access of replication factors to nucleosomal DNA. Thus, more information on the possible interaction between Pol subunits and proteins including histone acetyltransferases will provide insight into chromosomal DNA replication and the role of Pol in that process.
Recently Li et al. identified and cloned the two small subunits (p17 and p12 polypeptides) of HeLa Pol and found that they are homologues of S.cerevisiae Dpb4p and Dpb3p, respectively (36). Furthermore, we detected two smaller subunits similar to those two subunits of human Pol in Xenopus egg Pol (T.Ohya, S.Waga and A.Sugino, unpublished results). Thus, it is highly possible that the subunit structure of Pol is well conserved from lower eukaryotes to higher eukaryotes including human cells.
In summary, this study shows that the fourth subunit of S.cerevisiae Pol complex, and the overall subunit structure of Pol, is conserved in eukaryotes. In addition, the results indicate that while Dpb4p is not essential for cell viability, it plays a crucial role, along with Dpb3p, in maintaining the subunit structure of Pol and may facilitate interaction of the Pol complex with other replication proteins.
|
ACKNOWLEDGEMENTS |
|---|
This work was supported by Grants-in-Aid for Scientific Research on Priority Area from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Mitsubishi Foundation to A.S.
|
FOOTNOTES |
|---|
* To whom correspondence should be addressed. Tel: +81 6 6879 8331; Fax: +81 6 6877 3584; Email: asugino@biken.osaka-u.ac.jp Present address: Satoko Maki, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama-cho, Ikoma, Nara 630-0101, Japan
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-
1 Sugino,A. (1995) Trends Biochem. Sci., 20, 319–323.[ISI][Medline]
2 Aparicio,O.M., Weinstein,D.M. and Bell,S.P. (1997) Cell, 91, 59–69.[ISI][Medline]
3 Masumoto,H., Sugino,A. and Araki,H. (2000) Mol. Cell. Biol., 20, 2809–2817.
4 Waga,S. and Stillman,B. (1998) Annu. Rev. Biochem., 67, 721–751.[ISI][Medline]
5 Burgers,P.M. (1998) Chromosoma, 107, 218–227.[ISI][Medline]
6 Hashimoto,K., Nakashima,N., Ohara,T., Maki,S. and Sugino,A. (1998) Nucleic Acids Res., 26, 477–485.
7 Gerik,K.J., Li,X., Pautz,A. and Burgers,P.M. (1998) J. Biol. Chem., 273, 19747–19755.
8 Zuo,S., Bermudez,V., Zhang,G., Kelman,Z. and Hurwitz,J. (2000) J. Biol. Chem., 275, 5153–5162.
9 Morrison,A., Bell,J., Kunkel,T.A. and Sugino,A. (1991) Proc. Natl Acad. Sci. USA, 88, 9473–9477.
10 Simon,M., Giot,L. and Faye,G. (1991) EMBO J., 10, 2165–2170.[ISI][Medline]
11 Hamatake,R.K., Hasegawa,H., Clark,A.B., Bebenek,K., Kunkel,T.A. and Sugino,A. (1990) J. Biol. Chem., 265, 4072–4083.
12 Araki,H., Hamatake,R.K., Johnston,L.H. and Sugino,A. (1991) Proc. Natl Acad. Sci. USA, 88, 4601–4605.
13 Burgers,P.M.J. (1991) J. Biol. Chem., 266, 22698–22706.
14 Maki,S., Hashimoto,K., Ohara,T. and Sugino,A. (1998) J. Biol. Chem., 273, 21332–21341.
15 Morrison,A., Araki,H., Clark,A.B., Hamatake,R.K. and Sugino,A. (1990) Cell, 62, 1143–1151.[ISI][Medline]
16 Morrison,A. and Sugino,A. (1994) Mol. Gen. Genet., 242, 289–296.[ISI][Medline]
17 Araki,H., Ropp,P.A., Johnson,A.L., Johnston,L.H., Morrison,A. and Sugino,A. (1992) EMBO J., 11, 733–740.[ISI][Medline]
18 Navas,T.A., Zhou,Z. and Elledge,S.J. (1995) Cell, 80, 29–39.[ISI][Medline]
19 Navas,T.A., Sanchez,Y. and Elledge,S.J. (1996) Genes Dev., 10, 2632–2643.
20 Budd,M.E. and Campbell,J.L. (1993) Mol. Cell. Biol., 13, 496–505.
21 Araki,H., Hamatake,R.K., Morrison,A., Johnson,A.L., Johnston,L.H. and Sugino,A. (1991) Nucleic Acids Res., 19, 4867–4872.
22 Araki,H., Leem,S.-H., Phongdara,A. and Sugino,A. (1995) Proc. Natl Acad. Sci. USA, 92, 11791–11795.
23 Kamimura,Y., Masumoto,H., Sugino,A. and Araki,H. (1998) Mol. Cell. Biol., 18, 6102–6109.
24 Kesti,T., Flick,K., Keranen,S., Syvaoja,J.E. and Wittenberg,C. (1999) Mol. Cell, 3, 679–685.[ISI][Medline]
25 Dua,R., Levy,D.L. and Campbell,J.L. (1999) J. Biol. Chem., 274, 22283–22288.
26 Sugino,A., Ohara,T., Sebastian,J., Nakashima,N. and Araki,H. (1998) Genes Cells, 3, 99–110.[Abstract]
27 Hubscher,U., Nasheuer,I. and Syvaoja,J.E. (2000) Trends Biochem. Sci., 25, 143–147.[ISI][Medline]
28 Kaiser,C., Michaelis,S. and Mitchell,A. (1994) Methods in Yeast Genetics. A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
29 Berben,G., Duont,J., Gilliquet,V., Bolle,P.A. and Hilger,F. (1991) Yeast, 7, 475–477.[ISI][Medline]
30 Paulovich,A.G., Margulies,R.U., Garvik,B.M. and Hartwell,L.H. (1997) Genetics, 145, 45–62.[Abstract]
31 Kitada,K., Johnston,L.H., Sugino,T. and Sugino,A. (1992) Genetics, 131, 21–29.[Abstract]
32 Thompson,J.D., Higgins,D.G. and Gibson,T.J. (1994) Comput. Appl. Biosci., 10, 19–29.
33 Arents,G. and Moudrianakis,E.N. (1993) Proc. Natl Acad. Sci. USA, 90, 10489–10493.
34 Baxevanis,A.D., Arents,G., Moudrianakis,E.N. and Landsman,D. (1995) Nucleic Acids Res., 23, 2685–2691.
35 Currie,R.A. (1998) J. Biol. Chem., 273, 1430–1434.
36 Li,Y., Pursell,Z. and Linn,S. (2000) J. Biol. Chem., 275, 23247–23252.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Jaszczur, K. Flis, J. Rudzka, J. Kraszewska, M. E. Budd, P. Polaczek, J. L. Campbell, P. Jonczyk, and I. J. Fijalkowska Dpb2p, a Noncatalytic Subunit of DNA Polymerase {varepsilon}, Contributes to the Fidelity of DNA Replication in Saccharomyces cerevisiae Genetics, February 1, 2008; 178(2): 633 - 647. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Dang, M. N. Kagalwala, and B. Bartholomew The Dpb4 Subunit of ISW2 Is Anchored to Extranucleosomal DNA J. Biol. Chem., July 6, 2007; 282(27): 19418 - 19425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Tsubota, R. Tajima, K. Ode, H. Kubota, N. Fukuhara, T. Kawabata, S. Maki, and H. Maki Double-stranded DNA Binding, an Unusual Property of DNA Polymerase {epsilon}, Promotes Epigenetic Silencing in Saccharomyces cerevisiae J. Biol. Chem., October 27, 2006; 281(43): 32898 - 32908. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Seki, M. Akita, Y. Kamimura, S. Muramatsu, H. Araki, and A. Sugino GINS Is a DNA Polymerase {epsilon} Accessory Factor during Chromosomal DNA Replication in Budding Yeast J. Biol. Chem., July 28, 2006; 281(30): 21422 - 21432. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Dornfeld and M. Johnson AP endonuclease deficiency results in extreme sensitivity to thymidine deprivation Nucleic Acids Res., November 27, 2005; 33(20): 6644 - 6653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Tackett, D. J. Dilworth, M. J. Davey, M. O'Donnell, J. D. Aitchison, M. P. Rout, and B. T. Chait Proteomic and genomic characterization of chromatin complexes at a boundary J. Cell Biol., April 11, 2005; 169(1): 35 - 47. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-G. Spiga and G. D'Urso Identification and cloning of two putative subunits of DNA polymerase epsilon in fission yeast Nucleic Acids Res., September 23, 2004; 32(16): 4945 - 4953. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kesti, W. H. McDonald, J. R. Yates III, and C. Wittenberg Cell Cycle-dependent Phosphorylation of the DNA Polymerase Epsilon Subunit, Dpb2, by the Cdc28 Cyclin-dependent Protein Kinase J. Biol. Chem., April 2, 2004; 279(14): 14245 - 14255. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. D. McConnell, M. E. Gelbart, and T. Tsukiyama Histone Fold Protein Dls1p Is Required for Isw2-Dependent Chromatin Remodeling In Vivo Mol. Cell. Biol., April 1, 2004; 24(7): 2605 - 2613. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Iida and H. Araki Noncompetitive Counteractions of DNA Polymerase {varepsilon} and ISW2/yCHRAC for Epigenetic Inheritance of Telomere Position Effect in Saccharomyces cerevisiae Mol. Cell. Biol., January 1, 2004; 24(1): 217 - 227. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Feng, L. Rodriguez-Menocal, G. Tolun, and G. D'Urso Schizosacchromyces pombe Dpb2 Binds to Origin DNA Early in S Phase and Is Required for Chromosomal DNA Replication Mol. Biol. Cell, August 1, 2003; 14(8): 3427 - 3436. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Asahara, Y. Li, J. Fuss, D. S. Haines, N. Vlatkovic, M. T. Boyd, and S. Linn Stimulation of human DNA polymerase {epsilon} by MDM2 Nucleic Acids Res., May 1, 2003; 31(9): 2451 - 2459. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Edwards, C. M. Li, D. L. Levy, J. Brown, P. M. Snow, and J. L. Campbell Saccharomyces cerevisiae DNA Polymerase {varepsilon} and Polymerase {sigma} Interact Physically and Functionally, Suggesting a Role for Polymerase {varepsilon} in Sister Chromatid Cohesion Mol. Cell. Biol., April 15, 2003; 23(8): 2733 - 2748. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Shimizu, K. Hashimoto, J. M. Kirchner, W. Nakai, H. Nishikawa, M. A. Resnick, and A. Sugino Fidelity of DNA Polymerase epsilon Holoenzyme from Budding Yeast Saccharomyces cerevisiae J. Biol. Chem., September 27, 2002; 277(40): 37422 - 37429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ohya, Y. Kawasaki, S.-I. Hiraga, S. Kanbara, K. Nakajo, N. Nakashima, A. Suzuki, and A. Sugino The DNA Polymerase Domain of polepsilon Is Required for Rapid, Efficient, and Highly Accurate Chromosomal DNA Replication, Telomere Length Maintenance, and Normal Cell Senescence in Saccharomyces cerevisiae J. Biol. Chem., July 26, 2002; 277(31): 28099 - 28108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Dua, D. L. Levy, C. M. Li, P. M. Snow, and J. L. Campbell In Vivo Reconstitution of Saccharomyces cerevisiae DNA Polymerase epsilon in Insect Cells. PURIFICATION AND CHARACTERIZATION J. Biol. Chem., March 1, 2002; 277(10): 7889 - 7896. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Fuss and S. Linn Human DNA Polymerase epsilon Colocalizes with Proliferating Cell Nuclear Antigen and DNA Replication Late, but Not Early, in S Phase J. Biol. Chem., March 1, 2002; 277(10): 8658 - 8666. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||