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

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.

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Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5724-5735.1992 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5724-5735

Author(s):

J Miles ◽

T Formosa

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Polymerase ◽

Yeast Cells ◽

Chromosome Loss ◽

Dna Metabolism ◽

Checkpoint Gene ◽

Dna Polymerase Alpha ◽

In Cells

Potential DNA replication accessory factors from the yeast Saccharomyces cerevisiae have previously been identified by their ability to bind to DNA polymerase alpha protein affinity matrices (J. Miles and T. Formosa, Proc. Natl. Acad. Sci. USA 89:1276-1280, 1992). We have now used genetic methods to characterize the gene encoding one of these DNA polymerase alpha-binding proteins (POB1) to determine whether it plays a role in DNA replication in vivo. We find that yeast cells lacking POB1 are viable but display a constellation of phenotypes indicating defective DNA metabolism. Populations of cells lacking POB1 accumulate abnormally high numbers of enlarged large-budded cells with a single nucleus at the neck of the bud. The average DNA content in a population of cells lacking POB1 is shifted toward the G2 value. These two phenotypes indicate that while the bulk of DNA replication is completed without POB1, mitosis is delayed. Deleting POB1 also causes elevated levels of both chromosome loss and genetic recombination, enhances the temperature sensitivity of cells with mutant DNA polymerase alpha genes, causes increased sensitivity to UV radiation in cells lacking a functional RAD9 checkpoint gene, and causes an increased probability of death in cells carrying a mutation in the MEC1 checkpoint gene. The sequence of the POB1 gene indicates that it is identical to the CTF4 (CHL15) gene identified previously in screens for mutations that diminish the fidelity of chromosome transmission. These phenotypes are consistent with defective DNA metabolism in cells lacking POB1 and strongly suggest that this DNA polymerase alpha-binding protein plays a role in accurately duplicating the genome in vivo.

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Fe-S coordination defects in the replicative DNA polymerase delta cause deleterious DNA replication in vivo and subsequent DNA damage in the yeast Saccharomyces cerevisiae

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab124 ◽

2021 ◽

Author(s):

Roland Chanet ◽

Dorothée Baïlle ◽

Marie-Pierre Golinelli-Cohen ◽

Sylvie Riquier ◽

Olivier Guittet ◽

...

Keyword(s):

Dna Damage ◽

Cell Death ◽

Dna Replication ◽

Dna Polymerase ◽

Dna Polymerase Delta ◽

Chromosomal Dna ◽

Yeast Saccharomyces Cerevisiae ◽

C Terminus

Abstract B-type eukaryotic polymerases contain a [4Fe-4S] cluster in their C-terminus domain, whose role is not fully understood yet. Among them, DNA polymerase delta (Polδ) plays an essential role in chromosomal DNA replication, mostly during lagging strand synthesis. Previous in vitro work suggested that the Fe-S cluster in Polδ is required for efficient binding of the Pol31 subunit, ensuring stability of the Polδ complex. Here we analyzed the in vivo consequences resulting from an impaired coordination of the Fe-S cluster in Polδ. We show that a single substitution of the very last cysteine coordinating the cluster by a serine is responsible for the generation of massive DNA damage during S phase, leading to checkpoint activation, requirement of homologous recombination for repair, and ultimately to cell death when the repair capacities of the cells are overwhelmed. These data indicate that impaired Fe-S cluster coordination in Polδ is responsible for aberrant replication. More generally, Fe-S in Polδ may be compromised by various stress including anti-cancer drugs. Possible in vivo Polδ Fe-S cluster oxidation and collapse may thus occur, and we speculate this could contribute to induced genomic instability and cell death, comparable to that observed in pol3-13 cells.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.7884-7890.1994 ◽

1994 ◽

Vol 14 (12) ◽

pp. 7884-7890

Author(s):

M P Longhese ◽

P Plevani ◽

G Lucchini

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Dna Binding Protein ◽

Dna Polymerase Delta ◽

Gene Encoding ◽

Replication Factor ◽

Dna Polymerase Alpha ◽

Essential Step ◽

Primase Complex

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 RFA1 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 alpha-primase complex and with DNA polymerase delta.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.7884 ◽

1994 ◽

Vol 14 (12) ◽

pp. 7884-7890 ◽

Cited By ~ 79

Author(s):

M P Longhese ◽

P Plevani ◽

G Lucchini

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Dna Binding Protein ◽

Dna Polymerase Delta ◽

Gene Encoding ◽

Replication Factor ◽

Dna Polymerase Alpha ◽

Essential Step ◽

Primase Complex

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 RFA1 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 alpha-primase complex and with DNA polymerase delta.

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Characterization of Cdc47p-minichromosome maintenance complexes in Saccharomyces cerevisiae: identification of Cdc45p as a subunit.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5867 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5867-5875 ◽

Cited By ~ 42

Author(s):

S Dalton ◽

B Hopwood

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Replication ◽

Chromosomal Dna ◽

Minichromosome Maintenance ◽

Rate Limiting Step ◽

A Subunit

Cdc47p is a member of the minichromosome maintenance (MCM) family of polypeptides, which have a role in the early stages of chromosomal DNA replication. Here, we show that Cdc47p assembles into stable complexes with two other members of the MCM family, Cdc46p and Mcm3p. The assembly of Cdc47p into complexes with Cdc46p does not appear to be cell cycle regulated, making it unlikely that these interactions per se are a rate-limiting step in the control of S phase. Cdc45p is also shown to interact with Cdc47p in vivo and to be a component of high-molecular-weight MCM complexes in cell lysates. Like MCM polypeptides, Cdc45p is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae; however, Cdc45p remains in the nucleus throughout the cell cycle, whereas MCMs are nuclear only during G1. We characterize two mutations in CDC47 and CDC46 which arrest cells with unduplicated DNA as a result of single base substitutions. The corresponding amino acid substitutions in Cdc46p and Cdc47p severely reduce the ability of these polypeptides to assemble in a complex with each other in vivo and in vitro. This argues that assembly of Cdc47p into complexes with other MCM polypeptides is important for its role in the initiation of chromosomal DNA replication.

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Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5724 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5724-5735 ◽

Cited By ~ 66

Author(s):

J Miles ◽

T Formosa

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Polymerase ◽

Yeast Cells ◽

Chromosome Loss ◽

Dna Metabolism ◽

Checkpoint Gene ◽

Dna Polymerase Alpha ◽

In Cells

Potential DNA replication accessory factors from the yeast Saccharomyces cerevisiae have previously been identified by their ability to bind to DNA polymerase alpha protein affinity matrices (J. Miles and T. Formosa, Proc. Natl. Acad. Sci. USA 89:1276-1280, 1992). We have now used genetic methods to characterize the gene encoding one of these DNA polymerase alpha-binding proteins (POB1) to determine whether it plays a role in DNA replication in vivo. We find that yeast cells lacking POB1 are viable but display a constellation of phenotypes indicating defective DNA metabolism. Populations of cells lacking POB1 accumulate abnormally high numbers of enlarged large-budded cells with a single nucleus at the neck of the bud. The average DNA content in a population of cells lacking POB1 is shifted toward the G2 value. These two phenotypes indicate that while the bulk of DNA replication is completed without POB1, mitosis is delayed. Deleting POB1 also causes elevated levels of both chromosome loss and genetic recombination, enhances the temperature sensitivity of cells with mutant DNA polymerase alpha genes, causes increased sensitivity to UV radiation in cells lacking a functional RAD9 checkpoint gene, and causes an increased probability of death in cells carrying a mutation in the MEC1 checkpoint gene. The sequence of the POB1 gene indicates that it is identical to the CTF4 (CHL15) gene identified previously in screens for mutations that diminish the fidelity of chromosome transmission. These phenotypes are consistent with defective DNA metabolism in cells lacking POB1 and strongly suggest that this DNA polymerase alpha-binding protein plays a role in accurately duplicating the genome in vivo.

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In vitro replication of DNA containing either the SV40 or the polyoma origin

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1987.0071 ◽

1987 ◽

Vol 317 (1187) ◽

pp. 439-453 ◽

Cited By ~ 9

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Hela Cells ◽

Dna Binding Protein ◽

T Antigen ◽

Sv40 T Antigen ◽

Sv40 Origin ◽

Sv40 Dna ◽

Primase Complex

The replication of DNA containing either the polyoma or SV40 origin has been done in vitro . Each system requires its cognate large-tumour antigen (T antigen) and extracts from cells that support its replication in vivo . The host-cell source of DNA polymerase α - primase complex plays an important role in discriminating between polyoma T antigen and SV40 T antigen-dependent replication of their homologous DNA. The SV40 origin- and T antigen-dependent DNA replication has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, HeLa DNA polymerase α - primase complex, eukaryotic topoisomerase I and a single-strand DNA binding protein from HeLa cells are required. The latter activity, isolated solely by its ability to support SV40 DNA replication, sediments and copurifies with two major protein species of 72 and 76 kDa. Although crude fractions yielded closed circular monomer products, the purified system does not. However, the addition of crude fractions to the purified system resulted in the formation of replicative form I (RFI) products. We have separated the replication reaction with purified components into multiple steps. In an early step, T antigen in conjunction with a eukaryotic topoisomerase (or DNA gyrase) and a DNA binding protein, catalyses the conversion of a circular duplex DNA molecule containing the SV40 origin to a highly underwound covalently closed circle. This reaction requires the action of a helicase activity and the SV40 T antigen preparation contains such an activity. The T antigen associated ability to unwind DNA copurified with other activities intrinsic to T antigen (ability to support replication of SV40 DNA containing the SV40 origin, poly dT-stimulated ATPase activity and DNA helicase).

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A Coordinated Temporal Interplay of Nucleosome Reorganization Factor, Sister Chromatin Cohesion Factor, and DNA Polymerase α Facilitates DNA Replication

Molecular and Cellular Biology ◽

10.1128/mcb.24.21.9568-9579.2004 ◽

2004 ◽

Vol 24 (21) ◽

pp. 9568-9579 ◽

Cited By ~ 43

Author(s):

Yanjiao Zhou ◽

Teresa S.-F. Wang

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

S Phase ◽

Terminal Region ◽

Replication Complex ◽

Dna Polymerase Α ◽

Chromatid Cohesion ◽

Phase Progression

ABSTRACT DNA replication depends critically upon chromatin structure. Little is known about how the replication complex overcomes the nucleosome packages in chromatin during DNA replication. To address this question, we investigate factors that interact in vivo with the principal initiation DNA polymerase, DNA polymerase α (Polα). The catalytic subunit of budding yeast Polα (Pol1p) has been shown to associate in vitro with the Spt16p-Pob3p complex, a component of the nucleosome reorganization system required for both replication and transcription, and with a sister chromatid cohesion factor, Ctf4p. Here, we show that an N-terminal region of Polα (Pol1p) that is evolutionarily conserved among different species interacts with Spt16p-Pob3p and Ctf4p in vivo. A mutation in a glycine residue in this N-terminal region of POL1 compromises the ability of Pol1p to associate with Spt16p and alters the temporal ordered association of Ctf4p with Pol1p. The compromised association between the chromatin-reorganizing factor Spt16p and the initiating DNA polymerase Pol1p delays the Pol1p assembling onto and disassembling from the late-replicating origins and causes a slowdown of S-phase progression. Our results thus suggest that a coordinated temporal and spatial interplay between the conserved N-terminal region of the Polα protein and factors that are involved in reorganization of nucleosomes and promoting establishment of sister chromatin cohesion is required to facilitate S-phase progression.

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Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.57-66.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 57-66

Author(s):

M Zuber ◽

E M Tan ◽

M Ryoji

Keyword(s):

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Living Cells ◽

Nuclear Antigen ◽

Plasmid Replication ◽

Dna Polymerase Delta ◽

Dna Polymerase Alpha ◽

Proliferating Cell

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.

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