Mutations in conserved yeast DNA primase domains impair DNA replication in vivo.

Francesconi, Stefania; Longhese, Maria Pia; Piseri, Anna; Santocanale, Corrado; Lucchini, Giovanna; Plevani, Paolo

doi:10.1073/pnas.88.9.3877

Cited by 43 publications

(44 citation statements)

References 22 publications

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“…Strain TD-28 (A4Ta inol ura3-52 canl) has been previously described (29). Strains H1514 (MA Ta ura3-52 trpl-63 leu2-3,112) and H1515 (MA4Ta ura3-52 trpl-63 leu2-3,112) were constructed by A. M. Cigan in A. G. Hinnebusch's laboratory (National Institutes of Health, Bethesda, Md.).…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, poll, pnil, and pn2 conditional mutants are defective in premeiotic DNA synthesis and show an enhanced rate of intrachromosomal recombination and spontaneous mutation, which is generally correlated with the severity of their defects in cell growth and DNA synthesis (9,41,42). Since these mutant strains fail to accumulate high-molecular-weight DNA products (9,29), the formation of nicked and gapped replicated DNA molecules might be responsible for the hyperrecombination phenotype usually found in yeast DNA synthesis mutants (33).…”

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“…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 Cloning of the S. cerevisiae POLl, PRII, and PR12 genes, which encode the p180, p48, and p58 subunits of the yeast complex, respectively, and the study ofpoll, pril, and pn2 lethal and conditional alleles have been essential in establishing the roles of the corresponding gene products in mitotic DNA replication and in identifying functional domains in the polymerase and primase polypeptides (8,26,29,42,51,54). Moreover, poll, pnil, and pn2 conditional mutants are defective in premeiotic DNA synthesis and show an enhanced rate of intrachromosomal recombination and spontaneous mutation, which is generally correlated with the severity of their defects in cell growth and DNA synthesis (9,41,42).…”

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

Foiani¹,

Marini²,

Gamba³

et al. 1994

Mol. Cell. Biol.

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268

254

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The four-subunit DNA polymerase a-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 Cloning of the S. cerevisiae POLl, PRII, and PR12 genes, which encode the p180, p48, and p58 subunits of the yeast complex, respectively, and the study ofpoll, pril, and pn2 lethal and conditional alleles have been essential in establishing the roles of the corresponding gene products in mitotic DNA replication and in identifying functional domains in the polymerase and primase polypeptides (8,26,29,42,51,54). Moreover, poll, pnil, and pn2 conditional mutants are defective in premeiotic DNA synthesis and show an enhanced rate of intrachromosomal recombination and spontaneous mutation, which is generally correlated with the severity of their defects in cell growth and DNA synthesis (9,41,42). Since these mutant strains fail to accumulate high-molecular-weight DNA products (9, 29), the formation of nicked and gapped replicated DNA molecules might be responsible for the hyperrecombination phenotype usually found in yeast DNA synthesis mutants (33).No enzymatic activity has been found to be associated with the 86-kDa protein species (B subunit), which is tightly bound to the p180 polypeptide, and the physiological role of p86 is presently unknown (7). In vitro reconstitution studies with purified components indicate that p86 is not required for polymerase-primase interaction, and its presence does not change the catalytic properties of Pol a (6, 7). Recently, it has been proposed that the human p86 homolog might serve as a molecular tether during DNA replication, because this subunit mediates the in vitro interaction between the human Pol a-primase complex and T antigen bound to the SV40 origin of replication (14).As a first step in identifying the physiological role of the B subunit of the yeast Pol a-primase complex in vivo, we have mutagenized by the two-codon insertion mutagenesis method (3) the essential POL12 gene, which was recently found to encode the p86 polypeptide (7, 36a). The analysis of 923

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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

Foiani¹,

Marini²,

Gamba³

et al. 1994

Mol. Cell. Biol.

Self Cite

268

254

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“…In this case, RNA priming is essential for lagging-strand synthesis. Priming in eukaryotes is carried out by the primase activity of the RNA-primase-Pol-α complex 40,41 and is essential for lagging-strand synthesis, as well as for the initiation of leading-strand synthesis in normal DNA replication.Once the RNA primer is synthesized, it is extended by the Pol α generating a short DNA primer onto which the Pol δ system is assembled, similar to the leading-strand synthesis step (Fig. 2).…”

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Links between replication, recombination and genome instability in eukaryotes

Flores‐Rozas

Kolodner

2000

Trends in Biochemical Sciences

108

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Double-strand breaks in DNA can be repaired by homologous recombination including breakinduced replication. In this reaction, the end of a broken DNA invades an intact chromosome and primes DNA replication resulting in the synthesis of an intact chromosome. Break-induced replication has also been suggested to cause different types of genome rearrangements.The ability of cells to repair double-strand breaks (DSBs) is critical for cell viability. Failure to correct such damage can result in cell cycle arrest, cell death and, if repaired incorrectly, the loss of genetic information or the accumulation of mutations. DSBs can arise in cells by exposure to ionizing radiation 1 and other types of DNA-damaging agents or through mechanical stress. In addition, some cellular processes, like DNA replication, can generate DSBs. These could arise by the action of nucleases at unprotected sites where replication forks stall, as a consequence of DNA polymerases (Pols) replicating across nicked chromosomes, as well as by the active processing of stalled replication forks by specific enzyme systems [2][3][4] . DSBs can also occur naturally and play important roles in processes such as meiosis 5 , mating-type switching 6 and mammalian V(D)J recombination 7 .Cells have developed a number of mechanisms to repair such lesions. In Saccharomyces cerevisiae, most of the repair of DSBs is carried out by mechanisms that promote homologous recombination. This requires the existence in the cell of sequences that are homologous to those affected by the DSB. Non-homologous end-joining also rejoins DSBs in S. cerevisiae albeit less efficiently than homologous recombination; non-homologous endjoining appears to be of greater importance in mammalian cells 8 . General models of recombinationSeveral recent reviews have discussed recombination and the repair of DSBs in detail 4,9-14 . Given the increasing evidence that translocations and other types of genome rearrangements arise from inappropriate repair of DSBs, we will focus on the repair of DSBs by a homologous recombination pathway that involves extensive DNA synthesis. Such a process, termed break-induced replication (BIR), has been suggested based on the observations by 16 . In this study, activation of the transcription enhancer element HOT1 creates a recombination hotspot suggested to act by generating DSBs that are processed resulting in strains with distal markers converted to those present in the homologous chromosome. The resulting gene conversion, which could extend over regions as long as 75 kb, was proposed to arise by replication of a primer structure generated by the centromere-containing fragment invading the homologous chromosome in a process analogous to that described for bacteriophage T4 (Ref. 17 (Fig. 1a). In both cases the ends of the broken DNA molecule are degraded by exonucleases creating 3′ overhangs that invade the homologous template. The result is the formation of a D-loop in which the annealed invading end serves as a primer that is extended by the DNA syn...

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“…The N-terminal part of the large subunit (p58 N in human primase) provides a platform for interactions with p49 and Pol ␣ (5,16). Most structural elements contributing to NTP and DNA binding by the large subunit are located in the C-terminal half of the protein (p58 C in human primase) (11,(17)(18)(19)(20). The crystal structures of the * This work was supported, in whole or in part, by National Institutes of Health (NIH), NIGMS, Grant GM101167 (to T. H. T.) and NIH, NCI, Grant CA129925 (to Y. I. P.).…”

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