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The C-terminus of Dpb2 is required for interaction with Pol2 and for cell viability.

Isoz I, Persson U, Volkov K, Johansson E - Nucleic Acids Res. (2012)

Bottom Line: The dpb2-200 allele carried two mutations within the last 13 codons of the open reading frame, one of which resulted in a six amino acid truncation.This truncated Dpb2 subunit was co-expressed with Pol2, Dpb3 and Dpb4 in S. cerevisiae, but this Dpb2 variant did not co-purify with the other Pol ε subunits.In conclusion, the lack of Dpb2 did not appear to have a negative effect on Pol ε activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden.

ABSTRACT
DNA polymerase ε (Pol ε) participates in the synthesis of the leading strand during DNA replication in Saccharomyces cerevisiae. Pol ε comprises four subunits: the catalytic subunit, Pol2, and three accessory subunits, Dpb2, Dpb3 and Dpb4. DPB2 is an essential gene with unclear function. A genetic screen was performed in S. cerevisiae to isolate lethal mutations in DPB2. The dpb2-200 allele carried two mutations within the last 13 codons of the open reading frame, one of which resulted in a six amino acid truncation. This truncated Dpb2 subunit was co-expressed with Pol2, Dpb3 and Dpb4 in S. cerevisiae, but this Dpb2 variant did not co-purify with the other Pol ε subunits. This resulted in the purification of a Pol2/Dpb3/Dpb4 complex that possessed high specific activity and high processivity and holoenzyme assays with PCNA, RFC and RPA on a single-primed circular template did not reveal any defects in replication efficiency. In conclusion, the lack of Dpb2 did not appear to have a negative effect on Pol ε activity. Thus, the C-terminal motif of Dpb2 that we have identified may instead be required for Dpb2 to fulfill an essential structural role at the replication origin or at the replication fork.

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The isolated dpb2-200 and dpb-201 alleles cannot support growth. (A) The two alleles, dpb2-200 and dpb2-201, were tested for lethality and temperature sensitivity in plasmid shuffling experiments in E134-dpb2Δ cells. 5-FOA was used for selection against the wild-type URA3-containing pRS316-DPB2 plasmid. The pRS314-DPB2 plasmid with the TRP1 marker was the wild-type control that could grow on 5-FOA. Four clones of each transformant are presented. (B) Tetrad analysis of a DPB2/DPB2 homozygote and a DPB2/dpb2-200 heterozygote.
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gks880-F1: The isolated dpb2-200 and dpb-201 alleles cannot support growth. (A) The two alleles, dpb2-200 and dpb2-201, were tested for lethality and temperature sensitivity in plasmid shuffling experiments in E134-dpb2Δ cells. 5-FOA was used for selection against the wild-type URA3-containing pRS316-DPB2 plasmid. The pRS314-DPB2 plasmid with the TRP1 marker was the wild-type control that could grow on 5-FOA. Four clones of each transformant are presented. (B) Tetrad analysis of a DPB2/DPB2 homozygote and a DPB2/dpb2-200 heterozygote.

Mentions: We isolated lethal mutations in the DPB2 gene to examine the role of Dpb2 in vivo and its function in the Pol ε complex. DPB2 is an essential gene, so to screen for lethal mutations we constructed two separate low copy number plasmids that carried the wild-type DPB2 gene under the control of the wild-type DPB2 promoter. One plasmid carried TRP1 and the other carried URA3 as selectable markers, and the plasmid with TRP1 was mutagenized by treatment with hydroxylamine. Lethal mutations in the DPB2 gene were identified by plasmid shuffling between the wild type and mutagenized DPB2 genes in the E134-dpb2Δ yeast strain. That is, transformants carrying both the mutagenized plasmid and the DPB2 wild-type plasmid were first selected on SC-Ura-Trp plates followed by replica plating onto SC −Trp + 5-FOA to identify those unable to proliferate in the absence of the plasmid with both the URA3 gene and the wild-type DPB2 gene. These cells carried a potentially lethal mutation in the hydroxylamine-treated DPB2 gene. In the initial screen, 360 colonies were replica plated, and 11 of these were unable to grow on SC-Trp + 5-FOA. These 11 isolates were re-streaked and their inability to grow on 5-FOA was confirmed. The mutagenized plasmids were recovered from the yeast cells and the DPB2 gene was sequenced in both directions. We identified three separate plasmids that carried mutations in DPB2 and all three resulted in a truncated protein. Allele dpb2-198 carried a nonsense codon that led to a truncation at residue 437 and allele dpb2-199 carried three mutations (P592S, Q597stop and Q600stop) leading to a truncation at residue 597 (Table 1). One of the alleles, dpb2-200, carried two mutations located near the 3′-end of the DPB2 gene. These two mutations changed a proline to serine at amino acid 680 (P680S) and a glutamine to a nonsense codon at position 687 (Q687stop). The dpb2-200 allele was the most interesting allele because the two mutations were located within 13 residues of the C-terminus of the resulting protein and the truncation was only six residues. As the entire vector had been treated with hydroxylamine, we subcloned the dpb2-200 allele into an untreated pRS314 vector to confirm that the lethal phenotype was not due to any additional mutations in the vector backbone. We also tested whether the dpb2-200 allele was temperature sensitive and found that raising or lowering the temperature had no effect on viability (Figure 1A). Finally, we established a diploid strain that carried one dpb2-200 allele and one wild-type DPB2 allele. Tetrad analysis confirmed that dpb2-200 was a lethal mutation because only two viable spores were found in each of seven asci examined (Figure 1B). This was not an integration effect caused by the hygromycin resistance gene because integration of DPB2 using the same strategy gave four viable spores from each ascus (Supplementary Figures S2 and S3).Figure 1.


The C-terminus of Dpb2 is required for interaction with Pol2 and for cell viability.

Isoz I, Persson U, Volkov K, Johansson E - Nucleic Acids Res. (2012)

The isolated dpb2-200 and dpb-201 alleles cannot support growth. (A) The two alleles, dpb2-200 and dpb2-201, were tested for lethality and temperature sensitivity in plasmid shuffling experiments in E134-dpb2Δ cells. 5-FOA was used for selection against the wild-type URA3-containing pRS316-DPB2 plasmid. The pRS314-DPB2 plasmid with the TRP1 marker was the wild-type control that could grow on 5-FOA. Four clones of each transformant are presented. (B) Tetrad analysis of a DPB2/DPB2 homozygote and a DPB2/dpb2-200 heterozygote.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526264&req=5

gks880-F1: The isolated dpb2-200 and dpb-201 alleles cannot support growth. (A) The two alleles, dpb2-200 and dpb2-201, were tested for lethality and temperature sensitivity in plasmid shuffling experiments in E134-dpb2Δ cells. 5-FOA was used for selection against the wild-type URA3-containing pRS316-DPB2 plasmid. The pRS314-DPB2 plasmid with the TRP1 marker was the wild-type control that could grow on 5-FOA. Four clones of each transformant are presented. (B) Tetrad analysis of a DPB2/DPB2 homozygote and a DPB2/dpb2-200 heterozygote.
Mentions: We isolated lethal mutations in the DPB2 gene to examine the role of Dpb2 in vivo and its function in the Pol ε complex. DPB2 is an essential gene, so to screen for lethal mutations we constructed two separate low copy number plasmids that carried the wild-type DPB2 gene under the control of the wild-type DPB2 promoter. One plasmid carried TRP1 and the other carried URA3 as selectable markers, and the plasmid with TRP1 was mutagenized by treatment with hydroxylamine. Lethal mutations in the DPB2 gene were identified by plasmid shuffling between the wild type and mutagenized DPB2 genes in the E134-dpb2Δ yeast strain. That is, transformants carrying both the mutagenized plasmid and the DPB2 wild-type plasmid were first selected on SC-Ura-Trp plates followed by replica plating onto SC −Trp + 5-FOA to identify those unable to proliferate in the absence of the plasmid with both the URA3 gene and the wild-type DPB2 gene. These cells carried a potentially lethal mutation in the hydroxylamine-treated DPB2 gene. In the initial screen, 360 colonies were replica plated, and 11 of these were unable to grow on SC-Trp + 5-FOA. These 11 isolates were re-streaked and their inability to grow on 5-FOA was confirmed. The mutagenized plasmids were recovered from the yeast cells and the DPB2 gene was sequenced in both directions. We identified three separate plasmids that carried mutations in DPB2 and all three resulted in a truncated protein. Allele dpb2-198 carried a nonsense codon that led to a truncation at residue 437 and allele dpb2-199 carried three mutations (P592S, Q597stop and Q600stop) leading to a truncation at residue 597 (Table 1). One of the alleles, dpb2-200, carried two mutations located near the 3′-end of the DPB2 gene. These two mutations changed a proline to serine at amino acid 680 (P680S) and a glutamine to a nonsense codon at position 687 (Q687stop). The dpb2-200 allele was the most interesting allele because the two mutations were located within 13 residues of the C-terminus of the resulting protein and the truncation was only six residues. As the entire vector had been treated with hydroxylamine, we subcloned the dpb2-200 allele into an untreated pRS314 vector to confirm that the lethal phenotype was not due to any additional mutations in the vector backbone. We also tested whether the dpb2-200 allele was temperature sensitive and found that raising or lowering the temperature had no effect on viability (Figure 1A). Finally, we established a diploid strain that carried one dpb2-200 allele and one wild-type DPB2 allele. Tetrad analysis confirmed that dpb2-200 was a lethal mutation because only two viable spores were found in each of seven asci examined (Figure 1B). This was not an integration effect caused by the hygromycin resistance gene because integration of DPB2 using the same strategy gave four viable spores from each ascus (Supplementary Figures S2 and S3).Figure 1.

Bottom Line: The dpb2-200 allele carried two mutations within the last 13 codons of the open reading frame, one of which resulted in a six amino acid truncation.This truncated Dpb2 subunit was co-expressed with Pol2, Dpb3 and Dpb4 in S. cerevisiae, but this Dpb2 variant did not co-purify with the other Pol ε subunits.In conclusion, the lack of Dpb2 did not appear to have a negative effect on Pol ε activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden.

ABSTRACT
DNA polymerase ε (Pol ε) participates in the synthesis of the leading strand during DNA replication in Saccharomyces cerevisiae. Pol ε comprises four subunits: the catalytic subunit, Pol2, and three accessory subunits, Dpb2, Dpb3 and Dpb4. DPB2 is an essential gene with unclear function. A genetic screen was performed in S. cerevisiae to isolate lethal mutations in DPB2. The dpb2-200 allele carried two mutations within the last 13 codons of the open reading frame, one of which resulted in a six amino acid truncation. This truncated Dpb2 subunit was co-expressed with Pol2, Dpb3 and Dpb4 in S. cerevisiae, but this Dpb2 variant did not co-purify with the other Pol ε subunits. This resulted in the purification of a Pol2/Dpb3/Dpb4 complex that possessed high specific activity and high processivity and holoenzyme assays with PCNA, RFC and RPA on a single-primed circular template did not reveal any defects in replication efficiency. In conclusion, the lack of Dpb2 did not appear to have a negative effect on Pol ε activity. Thus, the C-terminal motif of Dpb2 that we have identified may instead be required for Dpb2 to fulfill an essential structural role at the replication origin or at the replication fork.

Show MeSH
Related in: MedlinePlus