Overproduction and functional analysis of DNA primase subunits from yeast and mouse

Santocanale, Corrado; Locati, F.; Muzi-Falconi, Marco; Piseri, Anna; Tseng, Ben; Lucchini, Giovanna; Plevani, Paolo

doi:10.1016/0378-1119(92)90396-7

Cited by 9 publications

(4 citation statements)

References 27 publications

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“…Bottom, aliquots of the cell culture (50 ml) were taken at the indicated times and protein extracts were prepared using non-denaturing conditions (see "Experimental Procedures"). For each sample, 2 mg of total protein were immunoprecipitated and analyzed by SDS-PAGE electrophoresis and Western blotting with monoclonal or affinity-purified polyclonal antibodies against the individual pol ␣-primase subunits (Plevani et al, 1985;Santocanale et al, 1992;Foiani et al, 1994). ated p86 is present (maternal complex), while two complexes, containing either the p86 or p91 forms of B subunit, can be detected in S phase (45 min after ␣-factor release) and they persist until mitosis. Moreover, the appearance of phosphorylated p91 associated with the other subunits of the complex is concomitant with the timing of B subunit phosphorylation observed by Western blot analysis performed on crude extracts before immunoprecipitation (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Phosphorylation of the DNA Polymerase -Primase B Subunit Is Dependent on Its Association with the p180 Polypeptide

Ferrari

Lucchini

Plevani

et al. 1996

Journal of Biological Chemistry

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The B subunit of the DNA polymerase (pol) ␣-primase complex executes an essential role at the initial stage of DNA replication in Saccharomyces cerevisiae and is phosphorylated in a cell cycle-dependent manner. In this report, we show that the four subunits of the yeast DNA polymerase ␣-primase complex are assembled throughout the cell cycle, and physical association between newly synthesized pol ␣ (p180) and unphosphorylated B subunit (p86) occurs very rapidly. Therefore, B subunit phosphorylation does not appear to modulate p180⅐p86 interaction. Conversely, by depletion experiments and by using a yeast mutant strain, which produces a low and constitutive level of the p180 polypeptide, we found that formation of the p180⅐p86 subcomplex is required for B subunit phosphorylation.

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Section: Resultsmentioning

confidence: 99%

Phosphorylation of the DNA Polymerase -Primase B Subunit Is Dependent on Its Association with the p180 Polypeptide

Ferrari

Lucchini

Plevani

et al. 1996

Journal of Biological Chemistry

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“…To further understand the functions and interactions of the complex, we are now using these mutants to search for interacting mutations in other genes. We have first focused our efforts on the p48 primase subunit, which appears to be a major determinant for the species specificity of DNA replication (5,51), and it has been shown to interact with RPA (13,34) as well as with polyomavirus T antigen (5). Yeast p48 is encoded by the PRI1 gene (35), and its amino acid sequence is highly conserved in the human, mouse, and Drosophila homologs (3,40,55).…”

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Yeast pip3/mec3 Mutants Fail To Delay Entry into S Phase and To Slow DNA Replication in Response to DNA Damage, and They Define a Functional Link between Mec3 and DNA Primase

Longhese

Fraschini

Plevani

et al. 1996

Molecular and Cellular Biology

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The catalytic DNA primase subunit of the DNA polymerase alpha-primase complex is encoded by the essential PRI1 gene in Saccharomyces cerevisiae. To identify factors that functionally interact with yeast DNA primase in living cells, we developed a genetic screen for mutants that are lethal at the permissive temperature in a cold-sensitive pril-2 genetic background. Twenty-four recessive mutations belonging to seven complementation groups were identified. Some mutants showed additional phenotypes, such as increased sensitivity to UV irradiation, methyl methanesulfonate, and hydroxyurea, that were suggestive of defects in DNA repair and/or checkpoint mechanisms. We have cloned and characterized the gene of one complementation group, PIP3, whose product is necessary both for delaying entry into S phase or mitosis when cells are UV irradiated in G1 or G2 phase and for lowering the rate of ongoing DNA synthesis in the presence of methyl methanesulfonate. PIP3 turned out to be the MEC3 gene, previously identified as a component of the G2 DNA damage checkpoint. The finding that Mec3 is also required for the G1- and S-phase DNA damage checkpoints, together with the analysis of genetic interactions between a mec3 null allele and several conditional DNA replication mutations at the permissive temperature, suggests that Mec3 could be part of a mechanism coupling DNA replication with repair of DNA damage, and DNA primase might be involved in this process.

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“…Nevertheless, yeast RF-A only partially substitutes for human RF-A in the in vitro replication of simian virus 40 (6), indicating that species-specific interactions between RF-A and other replication proteins are important for its biological activity. Similarly, the DNA polymerase ct (pol ao)-primase complex is involved in determining the species specificity of the DNA replication process (42,47,48). The RF-A and pol o-primase complexes are quite peculiar, since these two protein complexes are required during both the initiation and elongation steps of DNA replication (11,53,55,56).…”

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Replication factor A is required in vivo for DNA replication, repair, and recombination.

1994

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Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes. Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFAI gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae. This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination. Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase a-primase complex and with DNA polymerase 8.The identification of eukaryotic replication proteins has relied mainly on the use of in vitro replication systems. For example, the simian virus 40 system allowed the isolation of a number of replication proteins from human cell extracts and the reconstitution of an in vitro replication system with purified components (10,31,52,55). However, the answer to the question of whether these proteins function in vivo to replicate chromosomal DNA requires the production of mutations in the corresponding genes and the characterization of the obtained mutant strains. The use of the yeast Saccharomyces cerevisiae has been particularly useful for this purpose (8,9).The human single-stranded-DNA-binding protein (also known as replication protein A or replication factor A [RF-A]), originally identified as a protein factor required for simian virus 40 DNA replication in vitro, is a heterotrimer of 70-, 34-, and 11-kDa subunits (19,57,58). The p70 polypeptide is sufficient for DNA binding (6,32,59), while the function of the p34 and p1l subunits is still unknown. The p34 polypeptide is phosphorylated in a cell cycle-dependent manner (14,17,20,22), suggesting that RF-A might be a target of the regulatory mechanisms driving the G1-to-S phase transition during the cell cycle.The RF-A complex is highly conserved in all eukaryotes (6,39,58), and the three RF-A genes in budding yeast cells are essential for cell viability (7). Nevertheless, yeast RF-A only partially substitutes for human RF-A in the in vitro replication of simian virus 40 (6), indicating that species-specific interactions between RF-A and other replication proteins are important for its biological activity. Similarly, the DNA polymerase ct (pol ao)-primase complex is involved in determining the species specificity of the DNA replication process (42,47,48). The RF-A and pol o-primase complexes are quite peculiar, since these two protein complexes are required during both the initiation and elongation steps of DNA replication (11,53,55,56). In vitro studies indicate that they interact with each other as well as with T antigen (15,16,37,41,43), suggesting a pivotal role for these proteins in DNA replication. Moreover, the function of the RF-A complex might not be limited to the replication process, since human RF-A seems to be required for the nucleotide excision repair of UV-damaged DNA in vitro (12), and both yeas...

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Overproduction and functional analysis of DNA primase subunits from yeast and mouse

Cited by 9 publications

References 27 publications

Phosphorylation of the DNA Polymerase -Primase B Subunit Is Dependent on Its Association with the p180 Polypeptide

Phosphorylation of the DNA Polymerase -Primase B Subunit Is Dependent on Its Association with the p180 Polypeptide

Yeast pip3/mec3 Mutants Fail To Delay Entry into S Phase and To Slow DNA Replication in Response to DNA Damage, and They Define a Functional Link between Mec3 and DNA Primase

Replication factor A is required in vivo for DNA replication, repair, and recombination.

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