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Cloning DPB3, the Gene Encoding the Third Subunit of DNA Polymerase II of Saccharomyces Cerevisiae

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Cloning DPB3, the Gene Encoding the Third Subunit of DNA Polymerase II of Saccharomyces Cerevisiae Nucleic Acids Research, Vol. 19, No. 18 4867-4872 Cloning DPB3, the gene encoding the third subunit of DNA polymerase II of Saccharomyces cerevisiae Hiroyuki Araki*, Robert K.Hamatake+, Alan Morrison, Anthony L.Johnson1, Leland H.Johnston1 and Akio Sugino Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA and 12Laboratory of Cell Propagation, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK Downloaded from https://academic.oup.com/nar/article/19/18/4867/1078430 by guest on 27 September 2021 Received July 1, 1991; Revised and Accepted August 15, 1991 EMBL accession no. X58500 ABSTRACT INTRODUCTION DNA polymerase II purified from Saccharomyces The yeast Saccharomyces cerevisiae has three distinct, essential cerevisiae contains polypeptldes with apparent nuclear DNA polymerases (1-4). DNA polymerase I is similar molecular masses of >200, 80, 34, 30 and 29 kDa, the in structure and activity to mammalian DNA polymerase a (5,6), two largest of which (subunlts A and B) are encoded which has been proposed to replicate the lagging strand of by the essential genes POL2 and DPB2. By probing a chromosomal DNA (7). DNA polymerase HI is similar to \gt11 expression library of yeast DNA with antiserum mammalian DNA polymerase 6 (8), which has been proposed against DNA polymerase II, we isolated a single gene, as the leading strand replicase (7). Although DNA polymerase DPB3, that encodes both the 34- and 30-kDa II was first described in 1970 (9-11), its structure and function polypeptides (subunit C and C). The nucleotide were until recently unknown. DNA polymerase II purified to sequence of DPB3 contained an open reading frame near-homogeneity contains polypeptides with apparent molecular encoding a 23-kDa protein, significantly smaller than masses, >200, 80, 34, 30 and 29 kDa (12). The genes encoding the observed molecular masses, 34- or 30-kDa, which the > 200-kDa catalytic subunit A and the 80-kDa subunit B have might represent post-translationally modified forms of been cloned (4,13). Both genes are essential for cell growth and the DPB3 product. The predicted amlno acid sequence their thermosensitive mutants exhibit defective DNA synthesis contained a possible NTP-binding motif and a at the restrictive temperature, suggesting that the DNA glutamate-rich region. A dpb3 deletion mutant (dpb3A) polymerase II holoenzyme participates directly in chromosomal was viable and yielded a DNA polymerase II lacking the DNA replication (4,13; and Araki et al., in preparation). Based 34- and 30-kDa polypeptides. dpb3A strains exhibited on these results we have proposed a new model for the replication an increased spontaneous mutation rate, suggesting of DNA by three DNA polymerases (4). In this model, DNA that the DPB3 product is required to maintain fidelity polymerase I initiates synthesis at the origin and synthesizes DNA of chromosomal replication. Since a fifth, 29-kDa primers on the lagging strand, DNA polymerase II elongates the polypeptide was present in DNA polymerase II leading strand, and DNA polymerase m elongates the lagging preparations from wild-type cell extracts throughout strand. Yeast DNA polymerase II shares some similarities with purification, the subunit composition appears to be A, mammalian DNA polymerase e (previously described as 62 B, C (or C and C) and D. The 5' nontranscribed region polymerase), an enzyme previously suggested to be involved in of DPB3 contained the Af/ul-related sequence ACGCGA, DNA repair in human cells (14). while the 0.9-kb DPB3 transcript accumulated In this report we describe the cloning, sequencing and genetic periodically during the cell cycle and peaked at the analysis of DPB3, the gene encoding the third subunit of DNA G1/S boundary. The level of DPB3 transcript thus polymerase II. We found that both the 34- and 30-kDa appears to be under the same cell cycle control as polypeptides of purified DNA polymerase II are from the same those of POL2 , DPB2 and other DNA replication genes. gene, DPB3. Since 30-kDa polypeptide seemed to be a proteolytic DPB3 was mapped to chromosome II, 30 cM distal to product of 34-kDa polypeptide and the 29-kDa polypeptide also hls7. appears to be a subunit of DNA polymerase II, DNA polymerase • To whom correspondence should be addressed al Department of Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka 565, Japan + Present address: Department of Virology, the Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08540, USA 4868 Nucleic Acids Research, Vol. 19, No. 18 II thus contains four subunits. DPB3 is dispensable for cell constructed by the one-step gene replacement method (22) with growth, but is required for normal fidelity of chromosome a specific fragment containing the disrupted gene, followed by replication, and the level of its transcript appears to be under spore dissection. Yeast transformation, and other materials and the same cell cycle control as those of POL2 (Araki et al., in methods, were as described previously (23,4,12,13). preparation), DPB2 (13) and other DNA replication genes (for reviews, see 15 and 16). Rabbit antibodies against yeast DNA polymerase II The mouse antiserum against yeast DNA polymerase II (12) did not recognize the 29-kDa polypeptide of DNA polymerase II (12). MATERIALS AND METHODS Therefore, we have tried to raise rabbit antiserum against DNA Bacterial and yeast strains polymerase n (12) as described (24). This rabbit antiserum recognizes not only the >200-kDa, 80-kDa, 34-kDa and 30-kDa Escherichia coli DH5a (17) was used for the propagation of polypeptides previously recognized by the mouse antiserum, but plasmids. Y1090 (18) and LE392 (17) were used for Xgtll and also the 29-kDa polypeptide (see Fig. 6) by Western blotting. XCharon28A phages, respectively. 5. cerevisiae strains were Downloaded from https://academic.oup.com/nar/article/19/18/4867/1078430 by guest on 27 September 2021 YHA1 (MATa/MATa leu2-3,-112/leu2-3,-l 12 Measurement of spontaneous mutation rates trpl-289/trpl-289 ura3-52/ura3-52 his7-2/+ +/canl) (4), BJ3501 (MATapep4::HIS3prbl-Al.6 his3-A200 ura3-52 canl In preliminary experiments, spontaneous reversion frequencies were monitored by a patch test, in which patches of cells (about gal7) (from E. W. Jones, Carnegie-Mellon University, PA), 2 L155-1B (19), YHA3 (MATa odeS Ieu2 met8-l trpl ura3-52), 10 cm ) grown on YPDA were replica plated onto omission CG379(MATa ade5 his7-2 Ieu2-3,112 ura3-52) (from C. Giroux, media and revertants counted after five to ten days. Patches of DPB3+ cells yielded an average of one Lys+ revertant, and 40 Wayn State University, MI), XS803-2C (MATa hisl-7hom3-10 1 leu2-3,112 ura3-52 canl), and AMY50-1B (MATa lysl-1 hisl-7 His" " revertants. Subsequently, spontaneous reversion rates were ade2-l hom3-10 Ieu2-1 ura3-52). quantitated using the Leningrad test (25). Yeast cells grown in YPDA medium to stationary phase were washed, suspended in 7 Yeast genomic libraries and plasmid DNAs water at densities of approximately lO^to-lO cells/ml, and applied to plates using a multipronged replicator. Five-to-six Xgtll and XCharon28A 5. cerevisiae genomic DNA libraries hundred compartments were replicated for each determination. were described previously (4,20). Plasmid pBS(SK + ) The plates contained synthetic medium either lacking or (Stratagene, La Jolla, CA) was used for subcloning and DNA containing a limiting amount of either lysine, adenine or histidine. sequencing. Plasmid pGB310 contains the 5. cerevisiae URA3 Limiting concentrations were 2 /ig/ml histidine, 5 /tg/ml lysine, gene, excisable by £coRI, BamYQ., HindUl, Smal, and Sad (obtained from C. Giroux). Biochemical and genetical methods X gtll-2 _ Antibody screening of Xgtl 1 yeast genomic library (18), screening of XCharon 28A library with a DNA probe (17), double-stranded DNA sequencing by dideoxy-termination method (21), Western and Northern blottings were described previously (4). Partial purification of DNA polymerase II activity was as described (4,12). Synchronization of yeast cells by mating pheromone a, feed-starve, and elutriator centrifugation and the preparation of pDPBJ-2 total RNA from the yeast cells followed by Northern blotting were described (19). The gene disruption mutants were E.B,H Subunit 1 kb Figure 2. Structure of the DPB3 gene and construction of the dpb3A gene disruption. Xgtl 1-2 is a representative of the positives after screening a Xgtl 1 yeast genomic DNA library with mouse antiserum against DNA polymerase II. XCharon28A # 1 was isolated from the XCharon28A yeast genomic DNA library by plaque hybridization with the 1.3-kb£coRI insert of Xgtl 1-2. For construction of dpb3A, the 3.8-kb BamHl-BstEQ fragment of XCharon28A # 1 was first ligated to the pBS(SK+) DNA treated with BamHl and Kpnl, the remaining ends were blunt-ended by T4 DNA polymerase and ligated by T4 DNA ligase. Then, the Figure 1. The purified DNA polymerase n (12) was separated by 4-20% 1.2-Kb EcoRI-//wdlII URA3 fragment from pGB310 plasmid was inserted into SDS-PAGE and transferred to a Immobilon membrane filter as published (12). the above plasmid DNA after 0.32 Kb EcoUl-Hindm and 0.22-Kb HimiUl The filter was incubated either with 1000-fold diluted mouse antiserum against fragments were removed (pDPB3-2). This pDPB3-2 was further digested with polymerase II (lane 3) or with the antiserum purified with antigens produced by Spel and BsiEU and was used for transformation of diploki strain (YHA1) to Xgtl 1-2 (lane 1) or Xgtl 1-7 (lane 2) clones. The immuno-rcacted polypeptides make the dpb3A disruptant.
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