scholarly journals Reconstitution of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex in vitro. The 86-kDa subunit facilitates but is not required for complex formation

1991 ◽  
Vol 266 (16) ◽  
pp. 10093-10098
Author(s):  
R.G. Brooke ◽  
L.B. Dumas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Complex Formation ◽  
Dna Polymerase ◽  
Protein Complex ◽  
Dna Primase

Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex

1992 ◽  
Vol 12 (1) ◽  
pp. 155-163 ◽  
Author(s):  
K Fien ◽  
B Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Factor C ◽  
Dna Polymerase Delta ◽  
Replication Complex ◽  
Leading Strand ◽  
Factor C ◽  
Sv40 Dna

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.

Mechanism of initiation of in vitro DNA synthesis by the immunopurified complex between yeast DNA polymerase I and DNA primase

1986 ◽  
Vol 161 (2) ◽  
pp. 435-440 ◽  
Author(s):  
Gianfranco BADARACCO ◽  
Paola VALSASNINI ◽  
Marco FOIANI ◽  
Roberta BENFANTE ◽  
Giovanna LUCCHINI ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerase I ◽  
Dna Primase ◽  
Polymerase I

scholarly journals The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication.

1994 ◽  
Vol 14 (2) ◽  
pp. 923-933 ◽  
Author(s):  
M Foiani ◽  
F Marini ◽  
D Gamba ◽  
G Lucchini ◽  
P Plevani
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Cell Viability ◽  
Dna Polymerase ◽  
Chromosomal Dna ◽  
Dna Polymerase Alpha ◽  
B Subunit ◽  
Primase Complex

The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.

Initiation, elongation and pausing of in vitro DNA synthesis catalyzed by immunopurified yeast DNA primase: DNA polymerase complex.

1985 ◽  
Vol 4 (5) ◽  
pp. 1313-1317 ◽  
Author(s):  
G. Badaracco ◽  
M. Bianchi ◽  
P. Valsasnini ◽  
G. Magni ◽  
P. Plevani
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Polymerase Complex ◽  
Dna Primase

Fidelity of DNA polymerase I and the DNA polymerase I-DNA primase complex from Saccharomyces cerevisiae

1989 ◽  
Vol 9 (10) ◽  
pp. 4447-4458
Author(s):  
T A Kunkel ◽  
R K Hamatake ◽  
J Motto-Fox ◽  
M P Fitzgerald ◽  
A Sugino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Spontaneous Mutation ◽  
Catalytic Subunit ◽  
Dna Polymerase I ◽  
Spontaneous Mutation Rate ◽  
Single Base ◽  
Dna Primase ◽  
Polymerase I ◽  
Primase Complex

We have determined the fidelity of DNA synthesis by DNA polymerase I (yPol I) from Saccharomyces cerevisiae. To determine whether subunits other than the polymerase catalytic subunit influence fidelity, we measured the accuracy of yPol I purified by conventional procedures, which yields DNA polymerase with a partially proteolyzed catalytic subunit and no associated primase activity, and that of yPol I purified by immunoaffinity chromatography, which yields polymerase having a single high-molecular-weight species of the catalytic subunit, as well as three additional polypeptides and DNA primase activity. In assays that score polymerase errors within the lacZ alpha-complementation gene in M13mp2 DNA, yPol I and the yPol I-primase complex produced single-base substitutions, single-base frameshifts, and larger deletions. For specific errors and template positions, the two forms of polymerase exhibited differences in fidelity that could be as large as 10-fold. Nevertheless, results for the overall error frequency and the spectrum of errors suggest that the yPol I-DNA primase complex is not highly accurate and that, just as for the polymerase alone, its fidelity is not sufficient to account for a low spontaneous mutation rate in vivo. The specificity data also suggest models to explain -1 base frameshifts in nonrepeated sequences and certain complex deletions by a direct repeat mechanism involving aberrant loop-back synthesis.

The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences

1989 ◽  
Vol 9 (11) ◽  
pp. 4706-4712
Author(s):  
A H Siddiqui ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Complex Formation ◽  
Binding Activity ◽  
Lacz Gene ◽  
Wild Type ◽  
Mobility Shift ◽  
Proline Utilization

The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.

Novel Procedure To Investigate the Effect of Phosphorylation on Protein Complex Formation in Vitro and in Cells†

Biochemistry ◽  
2008 ◽  
Vol 47 (7) ◽  
pp. 2153-2161 ◽  
Author(s):  
Makoto Rembutsu ◽  
Marc P. M. Soutar ◽  
Lidy Van Aalten ◽  
Robert Gourlay ◽  
C. James Hastie ◽  
...  
Keyword(s):  
Complex Formation ◽  
Protein Complex ◽  
Protein Complex Formation ◽  
In Cells

scholarly journals Fidelity of DNA polymerase I and the DNA polymerase I-DNA primase complex from Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (10) ◽  
pp. 4447-4458 ◽  
Author(s):  
T A Kunkel ◽  
R K Hamatake ◽  
J Motto-Fox ◽  
M P Fitzgerald ◽  
A Sugino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Spontaneous Mutation ◽  
Catalytic Subunit ◽  
Dna Polymerase I ◽  
Spontaneous Mutation Rate ◽  
Single Base ◽  
Dna Primase ◽  
Polymerase I ◽  
Primase Complex

We have determined the fidelity of DNA synthesis by DNA polymerase I (yPol I) from Saccharomyces cerevisiae. To determine whether subunits other than the polymerase catalytic subunit influence fidelity, we measured the accuracy of yPol I purified by conventional procedures, which yields DNA polymerase with a partially proteolyzed catalytic subunit and no associated primase activity, and that of yPol I purified by immunoaffinity chromatography, which yields polymerase having a single high-molecular-weight species of the catalytic subunit, as well as three additional polypeptides and DNA primase activity. In assays that score polymerase errors within the lacZ alpha-complementation gene in M13mp2 DNA, yPol I and the yPol I-primase complex produced single-base substitutions, single-base frameshifts, and larger deletions. For specific errors and template positions, the two forms of polymerase exhibited differences in fidelity that could be as large as 10-fold. Nevertheless, results for the overall error frequency and the spectrum of errors suggest that the yPol I-DNA primase complex is not highly accurate and that, just as for the polymerase alone, its fidelity is not sufficient to account for a low spontaneous mutation rate in vivo. The specificity data also suggest models to explain -1 base frameshifts in nonrepeated sequences and certain complex deletions by a direct repeat mechanism involving aberrant loop-back synthesis.

scholarly journals Catalytic Roles of Yeast GSK3β/Shaggy Homolog Rim11p in Meiotic Activation

Genetics ◽  
1999 ◽  
Vol 153 (3) ◽  
pp. 1145-1152
Author(s):  
Krishnamurthy Malathi ◽  
Yang Xiao ◽  
Aaron P Mitchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Complex Formation ◽  
Mutant Proteins ◽  
Tyrosine Residues ◽  
Low Level ◽  
Separable Functions

Abstract In Saccharomyces cerevisiae, many meiotic genes are activated by a heteromeric transcription factor composed of Ime1p and Ume6p. Ime1p-Ume6p complex formation depends upon the protein kinase Rim11p, which interacts with and phosphorylates both Ime1p and Ume6p in vitro. Rim11p may promote complex formation through its phosphorylation of Ime1p and Ume6p or simply through its interaction with both proteins. Here, we characterize mutant Ime1p derivatives that interact with Rim11p but are not phosphorylated in vitro. These mutant proteins are also defective in interaction with Ume6p. These results argue that Ime1p must be phosphorylated to interact with Ume6p. Our genetic observations suggest that Ime1p tyrosine residues are among the Rim11p phosphoacceptors, and we find that Ime1p reacts with an anti-phosphotyrosine antibody. Ime1p and Rim11p have been thought to act only through Ume6p, but we find that Ime1p and Rim11p promote meiosis at a very low level in the absence of Ume6p. A nonphosphorylatable mutant Ime1p derivative promotes sporulation through this Ume6p-independent pathway, as does a mutant Rim11p derivative that fails to interact with Ime1p. Therefore, Ime1p and Rim11p have two genetically separable functions in the sporulation program. However, catalytic activity of Rim11p is required for sporulation in the presence or absence of Ume6p.

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