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Identification of the Fifth Subunit of Saccharomyces Cerevisiae Replication Factor C Sonja L

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Identification of the Fifth Subunit of Saccharomyces Cerevisiae Replication Factor C Sonja L 4986-4991 Nucleic Acids Research, 1995, Vol. 23, No. 24 Q--::;/l 1995 Oxford University Press Identification of the fifth subunit of Saccharomyces cerevisiae Replication Factor C Sonja L. Gary and Peter M. J. Burgers* Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid, St Louis, MO 63110, USA Received October 3, 1995; Revised and Accepted October 26, 1995 ABSTRACT cloned using a combination of peptide sequence analysis and homology-based PCR (16-19). However, it is not yet known Yeast replication factor C (RF-C) is a multipolypeptide whether these five polypeptides are sufficient to reconstitute the complex required for chromosomal DNA replication. human RF-C complex. Previously the complex from yeast Previously this complex was known to consist of at (yRF-C) was known to contain at least four subunits of 100, 41, least four subunits. We here report the identification of 40 and 37 kDa (6,13,20). The small subunits of yeast and human a fifth RF-C subunit from Saccharomyces cerevisiae, RF-C show high sequence similarity, both among themselves and encoded by the RFC5 (YBRO810) gene. This subunit between species. With the exception of the 38 kDa subunit, each exhibits highest homology to the 38 kDa subunit (38%) human RF-C subunit appears to have a yeast homolog with which of human RF-C (activator 1). Like the other four RFC it shares particular homology. genes, the RFC5 gene Is essential for yeast viability, In this paper we describe the identification of a fifth RF-C indicating an essential function for each subunit. RFC5 subunit from Saccharomyces cerevisiae encoded by the RFC5 mRNA Is expressed at steady-state levels throughout gene, which shows extensive sequence similarity to the 38 kDa the mitotic cell cycle. Upon overexpression in Escheri- subunit of human RF-C. This subunit is essential for viability of chia coil Rfc5p has an apparent molecular mass of yeast. In addition, its overexpression, together with 41 kDa. Overproduction of RF-C activity in yeast is overexpression of the other four RFC genes, is necessary and dependent on overexpresslon of the RFC5 gene sufficient for overproduction of RF-C in yeast. Therefore, it together with overexpression of the RFC1-4 genes, appears that all ofthe yeast RFC genes have now been identified. indicating that the RFC5 gene product forms an integral subunit of this replication factor. MATERIALS AND METHODS INTRODUCTION Strains Escherichia coli strains used were DH5 and BL21(DE3)pLysS. DNA replication in the eukaryotic nucleus may require the Yeast strains used were FM113 (MATa, ura3-52, trpl-289, activities of three essential DNA polymerases, a, 8 and £ (for leu2-3,112, prbl-1122, prl407, pep4-3) (a gift from M.Johnston), recent reviews see 1,2). Additional replication factors are prototrophic diploid NCYC239, PY2 (MMa, leu2-3,112, ura3-52, required forprocessive DNA synthesis at the replication fork. The trpl D, cani) and its derivative PY61 (as PY2, but rfcS::hisG- proliferating cell nuclear antigen (PCNA) is the processivity URA3-hisG and containing complementing plasmid pBL605), factor for DNA polymerases 8 and E (3-6). PCNA is a W303 (MATa/MATa, ura3-1/ura3-1, his3-11,JSIhis3-J1,15, homotrimer with a subunit molecular weight of 29 kDa and is trpl-1ltrpl-1, leu2-31leu2-3, ade2-11ade2-1, canl-1OO/canl-100) highly conserved from yeast to mammalian cells. The crystal and its derivative PY62 (as W303, but RFC51rfcS::hisG-UR- structure ofyeast PCNA shows that the trimer forms a closed ring A3-hisG). Strains PY61 and PY62 were crated by integrative with the appropriate dimensions and electrostatic properties to transformation with the disruption plasmid pBL607, which was encircle double-stranded DNA and to interact with it using previously digested with MunI and partially with HinduI to yield a non-specific contacts (7). Processivity in DNA synthesis is 5184 bp firgment (21). Disruption was confimned by genomic achieved by protein-protein interactions between PCNA and the Southem analysis. Except for the specific modifications noted, all polymerase, thereby tethering the DNA polymerase at the primer yeast protocols and media were as described (22). terminus (8). Replication factor C (RF-C) is a multipolypeptide complex Plasmids which loads PCNA onto the template-primer junction in an ATP-dependent manner (9-13). Human RF-C, also called Complementing plasmid pBL605 was created by ligating a 1830 activator 1, consists ofa large subunit of 140 kDa and four smaller bp HindIII-SalI fragment containing the RFC5 gene subunits of 36-41 kDa (14,15). All five known genes have been (nt 3552-5382 of GenBank accession no. X78993) with a linker * To whom correspondence should be addressed Nucleic Acids Research, 1995, Vol. 23, No. 24 4987 EcoRI site added to the HindIll site into the EcoRI-Sall site of minimal complete medium containing 2% lactate, 3% glycerol and pRS314 (Bluescript, TRPI, CEN6, ARSH4). pBL606 was 0.1% glucose as carbon source. After overnight growth at 30°C generated by ligating a 2076 bp HindIII-Clal fragment 100 ml rich medium containing the same carbon source mixture (nt 3552-5628 of X78993) into the AccI-HindIll site of pUC19. was added and after 3 h at 30°C 4 g galactose were added and cell The 1830 bp HindIII-SalI fragment of pBL606 was inserted into growth continued for another 3 h. All breakage and chromatogra- the HindIII-Sall site of pRS316 (Bluescript, URA3, CEN6, phy steps were carried out at 0-40C. Cells were lysed with glass ARSH4) to generate complementing plasmid pBL608. The beads in buffer A (final concentrations 50 mM Tris-HCl, pH 7.8, disruption plasmid pBL607 was created by replacing the central 5% glycerol, 1 mM EDTA, 3 mM dithiothreitol, 2 jM pepstatin A, region of RFC5 (MluI-BglIl, nt 4142-4811 of X78993) in 2 jM leupeptin, 10 mM NaHSO3, 0.5 M NaCl) as described (6). plasmid pBL606 with the hisG-URA3-hisG cassette (21). The cleared lysate was diluted with buffer B (50 mM Tris-HCl, pH pBL609 was generated from a PCR fragment containing the 7.8, 10% glycerol, 1 mM EDTA, 3 mM dithiothreitol, 2 jiM entire RFC5 gene in which a BspHI site and a HindlIl site were pepstatin A, 2 jM leupeptin, 10 mM NaHSO3) to reduce the NaCl created at the 5'- and 3'-ends of the open reading frame concentration to 0.2 M and gently shaken for 1 h with 0.5 ml respectively. These sites were generated using oligonucleotide Affigel Blue. The matrix was then loaded onto a column, washed primers which maintained the integrity of the amino acid with 2 ml buffer B plus 0.2 M NaCl, 2 ml buffer B plus 0.3 M NaCl sequence. The PCR product was digested with BspHI and HindIll and eluted with 1 ml buffer B plus 1 M NaCl. A Western blot and ligated into the NcoI-HindIII site ofE.coli expression vector analysis showed that all RF-C cross-reacting material was in the pPY55, containing the bacteriophage T7 genelO promoter and 1 M fraction. RF-C activity was measured in a DNA polymerase leader sequence. The integrity of the insert was confirmed by 6 holoenzyme assay as described below. DNA sequence analysis. Oligonucleotides were synthesized by DNagency. pBL417 contains the RFCJ, RFC2, RFC3 and RFC4 MonoS FPLC genes, each one positioned under transcriptional control of the GALI-JO UAS, in a 2 gm based vector, and URA3 as selectable FM113 cells containing vector or plasmid pBL417 marker (Impellizzeri,K.J. and Burgers,P.M., manuscript in (RFC1-RFC4) were grown on a 2 1 scale as described above, preparation). pBL419 contains the RFC5 gene inserted as a resulting in a yield of 15 g wet weight cells each. After cell MslI-SalI fragment (nt 3985-5382 of X78993) into the ClaI breakage and chromatography over a 10 ml Affigel Blue column (filled)-SalI sites of pRS424-GAL (Bluescript, 2,um ori, TRPI, as described above, the 1 M NaCl eluate was dialyzed against GAL1-JO). All restriction enzymes were purchased from New buffer C (as buffer B, but 30 mM HEPES-NaOH, pH 7.4,0.01% England Biolabs. Nonidet P-40) until the conductivity had reached that of buffer B plus 100 mM NaCl. The enzyme fraction was loaded onto a 1 ml MonoS FPLC column (Pharmacia, Piscataway, NJ), washed with Overproduction of Rfc5p in E.coli 2 ml buffer C plus 100 mM NaCl and eluted with a 15 ml linear A single colony of BL21(DE3)pLysS containing plasmid gradient of 100-600 mM NaCl in buffer C. pBL609 was inoculated into 5 ml LB medium with ampicillin (50 jg/ml) and chloramphenicol (34 jig/ml) and grown at 37°C. DNA polymerase 6 holoenzyme assay Once the OD595 had reached 0.4-0.6 isopropyl-[-D-galactopyra- The standard 30,l reaction contained 40 mM Tris-HCl, pH 7.8, noside was added to a final concentration of 1 mM. Three hours 8 mM MgCl2, 0.2 mg/ml bovine serum albumin, 1 mM after induction the cells were harvested, resuspended in 100 jil dithiothreitol, 100 jM each ofdATP, dCTP and dGTP and 25 jM 50 mM Tris-HCl, pH 7.5, 10% sucrose and frozen at -70°C. [3H]dTTP (100 c.p.m./pmol dNTP), 50 mM NaCl, 0.5 mM ATP, Upon thawing an equal volume of 2x lysis buffer was added (100 100 ng singly primed single-stranded mpl8 DNA (0.04 pmol of mM Tris-HCl, pH 8.1, 4 mM EDTA, 0.4 mM EGTA, 2 jiM circles), 850 ng E.coli SSB, 100 ng PCNA, 10 ng polymerase 6 leupeptin, 2 jM pepstatin A, 10 mM sodium bisulfite, 6 mM and RF-C or 2 gl MonoS fractions.
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