Gene. 90 (1990) 99-104 Elsevier

99

G E N E 03566

N u c l e o t i d e sequence and c h a r a c t e r i z a t i o n o f temperature-sensitive poll mutants o f Sacchammyces cerevisiae (DNA polymerase; primase; pop-out recombination; gene conversion; recombinant DNA; cloning)

Giovanna Lucchini', Marco Muzi Falconi ", Antonella Pizzagam ", Andres Aguilera b, Hannah L. Klein b and Paolo Plevani" ° Dipartimento di Genetica • Biologia dei Microrganismi. Universit~ di Milano, Milan (Italy) and b Department of Biochemistry. New York University Medical Center and Kaplan Cancer Center, New York, N ¥ (U.S.A.) Tel. (212) 340-5 778 Received by S.G. Oliver: 18 December 1989 Revised: 4 January 1990 Accepted: l0 January 1990

SUMMARY

We have analyzed the effects of temperature-sensitivity (ts)-conferring mutations in the Saccharomyces cerevisiae DNA polymerase I-encoding gene on cell growth, in vivo DNA synthesis, intrachromosomal gene conversion and pop-out recombination. Also, we have identified the molecular defect responsible for the ts phenotyp¢. Two mutant alleles (cdcl 7-1, cdcl 7-2) were originally identified as call-cycle mutations, while a third mutation (hpr3) was found during a genetic screening for mutants with a hyper-recombination phenotype. Both cdcl 7-2 and hpr3 cells complete one round ofcell division and DHA replication after shift to nonpermissive temperature, before being arrested as dumbbell-shaped cells. Conversely, the cdcl 7-1 mutation immediately blocks growth and DNA synthesis at 37°C. No substantial difference was observed in the frequency of intrachromosomal gene conversion and pop-out recombination events, when hpr3 and cdclT-I were compared to the previously characterized poU-I mutant. These two frequencies were ten- to 30-fold above wild-type level at semipermissive temperature. In each mutant, a single bp substitution, causing the replacement of Gly residues by either Asp (cdcl 7-1, cdcl7.2) or Glu (hpr3) in yeast DNA polymerase I is responsible for the ts phenotype.

INTRODUCTION

The yeast POLl gene encodes the 180-kDa Poll polypeptide (Johnson et al., 1985; Lucchini et al., 1985; Pizzagalli et al., 1988), that is found to be associated in vivo with three other protein species, to form a DNA polymerase-primase Correspondence to: Dr. G. Lucchini, Dipartimento di Genetica e di Biologia dei Microrganismi, Via Celoria 26, 20133 Milan (Italy) Tel.(2)2663498; Fax (2)2604551. Abbreviations: aa, amino acid(s); bp, base pair(s); FOA, 5-fluoro-orotic acid; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonuclootide; ORF, open reading frame; Poll, DNA polymerase I; POLl, yeast gene encoding Poll; S., Sacchavomyces; SD, synthetic minimal medium (see Table !, footnote b); ts, temperature-sensitive; UV, ultraviolet; wt, wild type; YEPD, rich medium (see Table I, footnote b); [ ], denotes plasmid-carrier state. 0378-1119/90/$03.50© 1990Elsevier SciencePubfishcrsB.V.(BiomedicalDivision)

complex similar to that found in most eukaryotic organisms (Plevaniet al., 1988; Kaguni and Lehman, 1988; Campbell, 1986). DNA primase activity results from the interactions between the two minor polypeptides (58 and 48 kDa; Foiani et al., 1989), while no function has been found until now for the fourth protein species (75 kDa). Cloning of the POLl gene has shown that it is unique in the haploid yeast genome, its function is essential for cell viability (Johnson et al., 1985; Plevani et al., 1988), and the level of its transcript fluctuates during mitotic and meiotic cell cycles. An increase in the amount of POLl mRNA is also observed after UV irradiation of a cell culture (Johnston et al., 1987). Moreover, cloning and characterization of the eel-cycle gene CDCI7 have shown that it is in fact POLl (Carson, 1987). The C-terminal half of the protein shares large regions of homology with several viral DNA polymerases, yeast DNA

100

polymerase III (or 6) and with human DNA polymerase 0c (Pizzagalli et al., 1988; Wang eta]., 1989; Boulet et al., 1989), and it may be involved in the catalytic activity of the enzyme (Larder et al., 1987; Knopf and Weisshart, 1988). The homology with the human enzyme extends to sequences in the N-terminal region. The characterization of the poll-I ts allele, whose gene product carries an aa substitution in one ofthese regions (Pizzagalli et al., 1988; Plevani et al., 1988), results in impaired ability to form a stable DNA polymerase-primase complex (Lucchini et al., 1988), suggesting that the conserved sequences in the N-terminal half might be important for protein-protein interactions. The aim of the present study was the molecular and physiological characterization of the independent pollts mutants cdclT-1, cdc17-2 (Hartwell, 1973) and hpr3 (Aguilera and Klein, 1988) since they should provide further information about the several possible roles of Poll in DNA metabolism and the definition of different functional domains in the protein.

synthesis. No differences between wt and mutant cells were observed for any of the parameters analysed at 25°C and for RNA synthesis after a shift to 37°C (data not shown). On the contrary, as shown in Fig. 1, both growth rate and DNA synthesis are arrested in the mutants after a temperature shift, but with different kinetics. Both hpr3 and cdcl 7-2 cells complete one cell-division cycle and one round of

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RESULTS AND DISCUSSION



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cdci?-l, cdcl?-2 and Apt3 Mutants cdclT-1 and cdcl?-2 had been previously characterized in Hartwelrs laboratory as cell-cycle mutants affected in telomere elongation and mitotic recombination (Carson and Hartwell, 1985; Hartwell and Smith, 1985). In an independent search for mitotic hyper.recombination mutations, one ts mutant, Apt3, was recovered that was an allele of CDCI7 (AguUera and Klein, 1988). We confirmed that poU-I (Pizzagalli etal., 1988), cdclT.l, cdc17-2 and hpr3 are allelic by 'examining the phenotype of all pairwise combinations of doubly heterozygous diploids. The mutations failed to complement each other, indicating that they map to one complementation group. We were able to recover spontaneous wt recombinants from poil-1/cdclT-I and hpr3/cdclT-I heterozygous diploids and, at much lower frequency, also from poll - l / cdc l 7-2 and hpr3/ cdc l 7-2. In contrast, hpr 3 failed to recombine with poll-I even after UV irradiation of the heterozygous diploids. This suggests that the poll-1 and hpr3 mutations map close to each other while the cdcl 7 mutations map in different regions of the gene. Since Poll is expected to contain different functional domains, a comparison of the kinetics ofgrowth and DNA synthesis arrest after temperature shift could be informative about possible different functions impaired in these mutants. Cells exponentially growing at 25°C were labeled with [SH]uracil and then shifted at 37°C. Samples were taken at different times at both temperatures and tested for cell number and morphology, as well as for DNA and RNA

107

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Fig, i, Growth rate and DNA synthesis for wt and mutant cells. Exponentially 8rowing cell cultures (25°C) were supplemented with 7 ~Ci/ml of[$,5-3H]uracil (50 Ci/mmol, Amersham International).After an additional 2.5 h at the permissive temperature, cultures were shil~ed to 37°C. Cell number (A) was monitored using a light microscope with a B0rker chamber. To measure the incorporation of the labeled precursor into DNA (B), the initial sample (TI) was taken after ! h of incubation at 25°C and the results are given as increase in incorporation at the indicated times relative to the amount of incorporation in TI samples. RNA and DNA synthesis were monitored as described in Lucchini et al. (1988). Mutants hpr3, cdcl7-1 and cdcl?-2 were backcrossed twice with wt strains TD28 and DGL2-1C, to obtain the strains DGL278-ga, DGL273-3D and DGL277-SB used in this work. We found no significant variation among wt strains or among strains bearing the same poll allele.

I01

DNA replication before ending as dumbbell-shaped cells. Such a delayed arrest behavior is typical of assembly mutants and it is identical to the previously observed effect of the poll-I mutation (Lucchini etal., 1988). The cdcl 7-1 cells rapidly stop both cell growth and DNA synthesis and dumbbell-shaped cells immediately appear after a temperature shift. A quick-stop phenotype is usually seen for mutants affected in the catalytic activity ofessential enzymes. Poll activity was assayed in crude extracts prepared from cells derived from the above cultures (Lucchini etal., 1988). The three mutants showed a reduction in activity to approx. one third the wt level, whether extracts were assayed at 23°C or at 37°C and whether cells were grown only at 23 ° C or shifted to 37 ° C (data not shown). Increased lability of Poll in crude mutant extracts has been observed previously with pollts mutants (Lucchini et al., 1988; Budd and Campbell, 1987). Overproduction of the mutant gone products will be necessary for their purification and biochemical characterization. (b) Intraehromosomal mitotic recombination in different poll ts mutants An enhancement of homologous recombination in homozygous diploids has been reported for the cdcl 7-1 (Hartwell and Smith, 1985) and Apt3 (Aguilera and Klein, 1988) mutants. It was of interest to compare the effect of different poll alleles using a genetic system capable of measuring both gone conversion and 'pop-out' recombination events. The system we used is depicted in Fig. 2A for a heteroallelic duplication of the LEO2 gone with the URA3 marker located between the duplicated genes. An analogous duplication was constructed carrying two different A/s? alleles flanking the TRPI gone. Recombinants are scored as Lea + or His ÷ segregants. Marker loss events are scored as U r a segregants, selected on FOA medium. As there is no selection for Trp- segregants, these were not studied. Although the cdcl 7-1 mutation appears to alter the catalytic properties of Poll, while the Apt3 and poll-I mutations seem to affect its interactions with DNA primase, both intrachromosomal gene conversion and pop-out recombination are increased at sam|permissive temperature in strains carrying any one of the three alleles. As shown in Fig. 2B, the Leu + and His* recombination rate is enhanced ten- to 30-fold. A similar enhancement in Urapop-out recombination events is seen. The Leu + recombinants can be divided into two classes: Leu + Ura + recombinants, which we call gone-conversion events, and Leu + U r a - recombinants, which we call pop-out events. When the Leu + recombinants are scored for the Ura phenotype, a 1 : 1 Ura + : Ura- ratio is found in the wt strains. This remains unchanged in the mutant strain. These data show that recombination enhancement is not limited to

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genetic exchange between homologs and confirm that it is not specific to a particular poll allele.

(e) Nucleotidesequencedeterminationofhpr3,cdclT-I

and

cdc17-2 In vivo recombination analysis with yeast-integrating plasmids carrying partially overlapping segments of the POLl gene (Fig. 3A) was used to map the hpr3 and cdcl 7-2 mutations in the 872-bp EcoRl fragment spanning from position + 1429 to + 2301 with respect to the ATG. By the same method, the cdclT-I mutation was located in the 280-bp SstI-SalI fragment, between nt + 2464 and + 2744. The mutant sequences were cloned (Fig. 3B, legend), and centromeric plasmids carrying a functional allele of POLl

102

TABLE I

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Strain No. •

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le.7.112::URA3::leu2.k .hb3-513::TRPI:: ". M A T " leu2-112::URA$::leu2-k hb3.513::TRPI:: hLv.?-537 urn3-52 ¢rpl adel-lOl M A T h leu2-112::UR,43::leu2-k his3.513::TRPl:: hb3-537 ura3-$2 Upl adel-lOl M A T h ura3.$2 inol ~nl MAT,, met4 lysl MATs leu2.112::URA3::leu2-k A/s3-$13::TRPI:: h/s3-537 urn3-52 trpl ariel.101 hpr3 MAT" leu2.112::URA3::leu2-k Ais3-513::TRPI:: hLv3-$37 urn3.52 wpl adel-101 Apt3 MATs urn?-52 inol ¢anl poll.! MATs his7 cdel7-1 horny ural MATn his7 cdcl 7-2 leu2 ura3 trpl MAT" urn.Y-52inol lysl Apt3 MATa ura3-52 lysl cam edelT-! MATa urn3-52 lysl inol canl cdclT-2 h/s$-537 ura3-52 wpl adel-lOl

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v-pol ltl sS8 ~i/blILOSlxs~ Fig..3. Mapping of~r3, cd¢17.1 and cdcl7.2 mutations. (A) Restriction m[p of the POLl chromosomal locus. An open box indicates the 4404-bp POLl ORF. Lines I, 2 and 3 near the top depict the extent efthe 3120-bp HindIIl-$all, the 2114-bp gall-EcoRl and the 2648-bp EcoRI.BamHI wt segments ofthe POLl gene cloned into the yeast integrating plasmid YIp5 (Botstein et al., 1979) to obtain plasmids pCM 16, pGL72 and pCM450 respectively. To map the position of the mutations, indicated by an asterisk (Apt3), a star (cdcl7.2) and a triangle (cdclT.l) (for aa changes, see part ~), the plasmids were linearized with Sstl (pCM 16 and pCM45) or HpuI (pGL72) and used to transform the yeast strains DGL273.3D, DGL278.9A and DGL277.SB, using LiCI4reated cells (Ito et al,, 1983). For each transformation, 48 purified Ura* transformants, selected on SD-Ura plates at 23°C, were tested on YEPD at 23°C and 37eC. Ifthe plasmid contains a POL ! fragment covering the chromosomal poll muta. tion, integration at POLI can restore a full.length wt allele.Temperature. resistant transformants were obtained from hpr3 and cdcl 7.2 strains only with plasmid pCMI6, the 872-bp wt EcoR! fragment of which was not contained within the other plasmids. For cdelT.I, recombinants with plasmid pGL72 retained the ts property, while plasmid pCM45 gave rise to temperature-resistant tremsformants. Integration of plasmid pCMI6 resulted only in ts transformants, which, however, were able to produce several temperature.resistant papillae, when spotted on YEPD plates and incubated at 37°C. Some of these papillae were Urn + and some U r a ' . Such results are expected for a mutation located inside the 280-bp gstI-$all fragment, present in both I~M 16 and pCM45 plasmids, when integration is directed to the gstl site. (B) Sequences affected by the poll mutations. To clone the 3120-bp HIndlIi-$all POLl fragment containing the Apt3, ¢dc!7.2 or cdclT-I mutations, total DNA prepared from the transformant strains DGL278-9A[pCM16], DGL277-SB[pCMI6] and DGL273-3D[pCM16] was cut with Hlndlll (kpr3 and edcl7.2) or with galI (cdcl 7-I)~The digested DNA was then ligated and used to transform EscAertchla coil cells. The recovered plasmids pMMI, pMM2$ and pMMI3, when used to transform kpr3, cdc17-2 or cdciT.! ts strains, respectively, 8ave rise only to Ura* ts clones, as expected ifthey carried HbMIIl.$all POLl fragments containing the/~pr3, cdc!7.2 and cdcl7-1 mutations, respectively. The nt sequencing was performed by the dideoxy chain-termination method (Sanger et at., 1977). Synthetic oligos were used as primers to sequence, on both strands, the region between nt + 1400 and + 2400 from the POLl ATG, including the 872-bp EcoRl

TSI-4** HI7CI-BI H!7-2 DGL278-9A** DGL273-3D** DGL277-5B **

" Strains HITCI-BI and H17-2 were kindly provided by L.H. Hartwell (University of Washington, Seattle, WA). All the other strains are from H.L. Klein (*) or G. Lucchini (**) laboratories. b :: indicates that a copy of the UP,A? or TRPI genes is integrated between the leu2-112 and leu2-k or the h/s3-$13 and hb3-537 alleles, respectively. Rich medium (YEPD), synthetic medium (SD) and sporulation medium, used in this study, were prepared according to Sherman 41986). FOA was used at a final concentration of 750 ~g/ml.

were used to substitute the wt 872-bp EcoRI fragment with the one derived from either hpr3 or cdc17-2. Similarly, the 280-bp wt gstl-$all fragment was replaced with the corresponding edcl 7-1 segment. The three derived plasmids were used to transform a diploid strain heterozygous for a 2325-bp deletion in the POLl coding region. Sporulation and tetrad analysis showed that the plasmids were able to complement the lethal chromosomal mutation at 25 oC but not at 37°C (data not shown), thus confn'ming that the mutant sequences had been cloned and properly mapped. The nt sequence analysis (Fig. 3B) has shown that each mutant had suffered a single O: C ~ A: T transition responsible for the ts phenotype. As shown in Fig. 3C, each aa substitution is found in a region which is highly conserved in DNA polymerases. The hpr3 aa change affects the

fragment, in plasmids pMMI and pMM25, and the region between nt + 2300 and + 3000, including the 280-bp gstl-gall fragment, in plasmid pMMI3. (C) Homology with other DNA polymerasesofthe as sequences altered by the mutations. Dots denote identical residues. Region P is conserved only in human DNA polymerase ~ while regions IV and VI are conserved also in several viral DNA polymerases. The aa residues changed in the mutants are indicated by the same symbols as used in (A). Y, yeast; H, human.

I03

same residue altered in poll-1 (Pizzagalli et al., 1988) and falls in a region which is conserved only in yeast and human DNA polymerase 0c(Plevani et al., 1988). The cdcl 7-1 and cdcl 7-2 mutations map in two regions which share a high degree of homology with several DNA polymerases (Wang et al., 1989; Boulet et eLI., 1989), but their different effect suggests they might define distinct functional domains.

(d) Conelusions (1) The physiological and molecular characterization of poll ts mutants described in this paper supports the hypothesis of different functional domains in Poll. Although the consequence of an aa change in a mutant enzyme must be interpreted with some caution in the absence of a specific knowledge of its effect on the overall conformation of the active enzyme, we believe that sequence analysis of a number of mutant alleles, in conjunction with protein sequence comparison, might be highly informative. (2) The cdcl7-1 aa change maps in the C-terminal half of Poll, containing five almost contiguous regions conserved in several replicative DNA polymerases (called regions I, II, llI, V and VI) (Wang et al., 1989; Fig. 3C). The majority of mutations altering the sensitivity of viral DNA polymerases to dNTP analogs or pyrophosphate have been localized in these regions (Larder et al., 1987; Knopf and Weisshart, 1988) which are therefore considered as potential substrate-binding domains. Mapping of the quick-stop mutations poll.17 in region III (Budd et al., 1989) and cdcl 7-1 in region VI (this work) is consistent with the interpretation that a quite large portion of the C-terminal half of the enzyme is essential for catalytic activity. We favor a model in which interactions among the conserved regions determine the formation of the catalytic site. (3) The slow-stop phenotype of hpr3 and cdcl 7-2 mutants is suggestive of a defect in assembly of replication complexes, as already shown for the poll-I mutant (Lucchini et al., 1988). Indeed, the hpr3 mutation causes a different aa substitution at the same residue altered in poll- 1 (Pizzagaili et al., 1988), in region P (Fig. 3C), conserved only in human and yeast DNA polymerase ~ (or I) (Plevani et al., 1988; Wang et al., 1989), which are both associated with DNA-primase activity. These data suggest a role for region P in the formation of the DNA-primase interaction domain. The cdcl 7-2 mutation causes an aa substitution in region IV, the only one conserved in the N-terminal half of several DNA polymerases, that might be important for protein-protein interactions with a common component of the replication complex. (4) PolI does not appear to have a major role in mitotic recombination. In agreement with previous models (Hartwell and Smith, 1985), our data support the hypothesis that the action of a defective Poll on a DNA template will ultimately lead to a nicked or gapped substrate that is

recombinogenic. In these conditions, even if the enzyme is active in DNA repair, DNA lesiovs are_ so numerous that recombination is stimulated.

ACKNOWLEDGEMENTS

We thank Dr. L.H. H artwell (University of Washington, Seattle, WA) for providing yeast strains. This work was supported by NIEHS grant ESO3847 (H.L.K.) and by a grant from the Pmgetto F~alizzato Biotecnologie e Biostrumentazione, C.N.R., Rome, Italy.

REFERENCES Aguilera, A. and Klein, H.L.: Genetic control of intrachromosomal recombination, I. Isolation and genetic characterization of hyperrecombination mutations. Genetics ! 19 (1988) 779-790. Botstein, D., Falco, S.C., Steward, S.E., Brennan, M., Scherr, S., Stinchcomb, D.T., StruM, K. and Davis, R.W.: Sterile host yeast (SHY): a eukaryntic system of biological containment for recombinant DNA experiments. Gene 8 (19"/9) 17-24. Boulet, A., Simon, M., Faye, G., Bauer, G.A. and Burgers, P.MJ.: Structure and function of the Saeckaromyees cerevisiae CDC2 gene encoding the large subanit ofDNA polymarase Ill. EMBO J. 8 (1989) 1849-1854. Budd, M. and Campbell, J.L: Temperature-sensitive mutations in the yeast DNA polymerase ! gene. Proc. Natl. Acad. Sci. USA 84 (1987) 2838-2842. Budd, M.E., Winrup, K.D., Bailey, J.E. and Campbell, J.L.: DNA polymerase i is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces¢erevisiae.Mol. Cell. Biol. 9 (1989) 365-376. Campbell, J.L.: Eukaryotic DNA replication. Annu. Rev, Biechem. $5 (1986) 733-771. Carson, MJ.: CDC/7, the Structural Gene for DNA Polymerase I of Yeast: Mitotic Hyper-recombination and Effects on Telomere Metabolism. Ph. D. Thesis, University of Washington, Seattle, WA, 1987. Carson, M.J. and Hartwell, LH.: CDCI 7: an essential gene that prevents telomere elongation in yeast. Cell 42 (1983) 249-257. Foiani, M., Lindner, A.G., Hartmann, G.R., Luechini, G. and Plevani, P.: Affinitylabeling of the active center and ribonucleoside triphosphatebinding site of yeast DNA primase. J. Biol. Chem. 264 (1989) 2189-2194. Hartwell, L.H.: Three additional genes required for deoxyribonucleicacid synthesis in Saccharomyces cerevbiae. J. Bacteriol. il5 (1973) 966-974. Hartwell, L.H. and Smith D.: Altered fidelity of mitotic chromosome transmission in cell cycle mutants ofS. cerevbiae.Genetics 110(1985) 381-395. ito, H., Yukuda, Y., Murata, K. and Klmura, A.: Transformation ofintact yeast cells treated with alkali cations. J. Buctariol. 153 (1983) 163-168. Johnson, L.M., Snyder, M., Chang, L.M.S., Davis, R.W. and Campbell, J.L.: Isolation of the gene encoding yeast DNA polymerase I. Cell 43 (1985) 369-377. Johnston, L.H., White, J.H.M., Johnson, A.L., Lucchini, G. and Plevani, P.: The yeast DNA polymerase ! transcript is regulated in both the mitotic cell cycle and in meiosis and is also induced alter DNA damage. Nucleic Acids Res. 15 (1987) 5017-5030.

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Kagani, LS. and Lehman, I.R.: Eukaryotic DNA polymerase-primase: structure, mechanism and function. Biochim. Biophys. Acta 950 (1988) 87-101. Knopf, C.W. and Weisshart, K.: The Herpes s/mplex virus DNA polymerase: analysis of'the functional domains. Biochim. Biophys. Acta 951 (1988) 298-314. Lard~, B.A., Kemp, S.D. and Darby, (3.: Related functional domains in virus DNA polymerases. EMBO J. 6 (1987) 169-175. Lea, D.E. and Coalson, LA.: The distribution of'the numbers of'mutants in bacterial populations. J. Genct. 49 (1948) 264-284. Luechini, G., Brandazza, A., Badaracco, G., Bianchi, M. and Plevani, P.: Identification of the yeast DNA polymerase I gene with antibody probes. Curt. Genet. 10 (1985) 245-252. Lucchini, G., Mazza, C., Scacheri, E. and Plevani, P.: Genetic mapping of$. cerel~vlaeDNA polymerase I gene and characterizationof a poll temperature sensitive mutant altered in the DNA primasepolymerase complex stcbility, Mol. Gen. Genet. 212 (1988) 459-465.

Pizzagaili, A., Valsasnini, P., Plevani, P. and Lucchini, G.: DNA polymerase I gene of Saccharomyces cere1~fiae:nuclcotide sequence, mapping of a temperature-sensitive mutation and protein homology with other DNA polymerases. Proc. Natl. Acad. Sci. USA 85 (1988)

3772-3776. Plevani,P.,Foiani,M., Muzi Falconi,M., Pizzagalli,A., Santoennale,C., Francesconi, S., Valsasnini, P., Comedini, A., Piatti, S. and Lucchini, G.: The yeast DNA polymerase-primase complex: genes and proreins. Biochim. Biophys. Acta 951 (1988) 268-273. Sanger, F., Nicklen, S. and Coulsan, A.R.: DNA sequencing with chainterminating inhibitors. Proc~ Natl. Acad. Sci. USA 74 (1977) 5463-5467. Sherman, F., Fink, G.R. and Hicks, J.B.: Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1986. Wang, T.S.F., Won8, S.W. and Korn, D.: Human DNA polymerase ~,: predicted functional domains and relationships with viral DNA polymerases. FASEB J. 5 (1989) 14-21.

Nucleotide sequence and characterization of temperature-sensitive pol1 mutants of Saccharomyces cerevisiae.

We have analyzed the effects of temperature-sensitivity (ts)-conferring mutations in the Saccharomyces cerevisiae DNA polymerase I-encoding gene on ce...
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