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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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Co-expression of Dpb2-201 together with Pol2, Dpb3 and Dpb4. (A) Western-blot analysis of pY116 cells (16) that over-expressed Dpb2-201 or Dpb2 in the presence of galactose. A rabbit polyclonal antibody was used that recognizes Dpb2. Two isolates carrying either over-expression plasmids with dpb2-201 or DPB2 were analyzed. An equal amount of cells were loaded in each lane after boiling in a loading buffer with SDS, β-mercaptoethanol, bromophenol blue and glycerol. The first four lanes (1–4) are cells harvested before induction with galactose and the next four lanes (5–8) are cells harvested after induction with galactose. Lanes labeled A, B, C and D are the same cultures before and after induction, respectively. Fifteen nanograms, 30 ng and 60 ng of purified Pol ε were loaded as indicated in the last three lanes (9–11). (B) The purified Pol ε complex was missing the Dpb2 subunit when purified from cells with galactose-induced over-expression of Dpb2-201 (Figure 2A). The estimated subunit stoichiometry of the purified Pol2/Dpb3/Dpb4 complex was 1:1.1:1.4 based on a plot of the density of the Coomassie-stained bands.
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gks880-F2: Co-expression of Dpb2-201 together with Pol2, Dpb3 and Dpb4. (A) Western-blot analysis of pY116 cells (16) that over-expressed Dpb2-201 or Dpb2 in the presence of galactose. A rabbit polyclonal antibody was used that recognizes Dpb2. Two isolates carrying either over-expression plasmids with dpb2-201 or DPB2 were analyzed. An equal amount of cells were loaded in each lane after boiling in a loading buffer with SDS, β-mercaptoethanol, bromophenol blue and glycerol. The first four lanes (1–4) are cells harvested before induction with galactose and the next four lanes (5–8) are cells harvested after induction with galactose. Lanes labeled A, B, C and D are the same cultures before and after induction, respectively. Fifteen nanograms, 30 ng and 60 ng of purified Pol ε were loaded as indicated in the last three lanes (9–11). (B) The purified Pol ε complex was missing the Dpb2 subunit when purified from cells with galactose-induced over-expression of Dpb2-201 (Figure 2A). The estimated subunit stoichiometry of the purified Pol2/Dpb3/Dpb4 complex was 1:1.1:1.4 based on a plot of the density of the Coomassie-stained bands.

Mentions: To determine whether the lethal phenotype was due to a non-functional Pol ε, we created an allele called dpb2-201 that was identical to dpb2-200 except that the five codons after the Q687stop mutation were removed. This was done to minimize the risk of accidentally reading through the stop codon during translation. We first confirmed that the dpb2-201 allele was lethal in the low copy vector pRS314–dpb2-201 (Figure 1A). Next, we cloned the dpb2-201 allele into an over-expression vector under the control of the galactose-inducible promoter (plasmid pJL9-201) and co-expressed Dpb2-201 together with Pol2, Dpb3 and Dpb4. The Dpb2-201 protein was detected in a western blot of cell extracts obtained after induction with galactose (Figure 2A). Next, we applied a previously developed purification protocol for Pol ε that depends on ion-exchange chromatography and size-exclusion chromatography and found that Dpb2-201 did not co-purify with the other subunits (Figure 2B). This suggested that Dpb2-201 was unable to interact with the other Pol ε subunits.Figure 2.


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)

Co-expression of Dpb2-201 together with Pol2, Dpb3 and Dpb4. (A) Western-blot analysis of pY116 cells (16) that over-expressed Dpb2-201 or Dpb2 in the presence of galactose. A rabbit polyclonal antibody was used that recognizes Dpb2. Two isolates carrying either over-expression plasmids with dpb2-201 or DPB2 were analyzed. An equal amount of cells were loaded in each lane after boiling in a loading buffer with SDS, β-mercaptoethanol, bromophenol blue and glycerol. The first four lanes (1–4) are cells harvested before induction with galactose and the next four lanes (5–8) are cells harvested after induction with galactose. Lanes labeled A, B, C and D are the same cultures before and after induction, respectively. Fifteen nanograms, 30 ng and 60 ng of purified Pol ε were loaded as indicated in the last three lanes (9–11). (B) The purified Pol ε complex was missing the Dpb2 subunit when purified from cells with galactose-induced over-expression of Dpb2-201 (Figure 2A). The estimated subunit stoichiometry of the purified Pol2/Dpb3/Dpb4 complex was 1:1.1:1.4 based on a plot of the density of the Coomassie-stained bands.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gks880-F2: Co-expression of Dpb2-201 together with Pol2, Dpb3 and Dpb4. (A) Western-blot analysis of pY116 cells (16) that over-expressed Dpb2-201 or Dpb2 in the presence of galactose. A rabbit polyclonal antibody was used that recognizes Dpb2. Two isolates carrying either over-expression plasmids with dpb2-201 or DPB2 were analyzed. An equal amount of cells were loaded in each lane after boiling in a loading buffer with SDS, β-mercaptoethanol, bromophenol blue and glycerol. The first four lanes (1–4) are cells harvested before induction with galactose and the next four lanes (5–8) are cells harvested after induction with galactose. Lanes labeled A, B, C and D are the same cultures before and after induction, respectively. Fifteen nanograms, 30 ng and 60 ng of purified Pol ε were loaded as indicated in the last three lanes (9–11). (B) The purified Pol ε complex was missing the Dpb2 subunit when purified from cells with galactose-induced over-expression of Dpb2-201 (Figure 2A). The estimated subunit stoichiometry of the purified Pol2/Dpb3/Dpb4 complex was 1:1.1:1.4 based on a plot of the density of the Coomassie-stained bands.
Mentions: To determine whether the lethal phenotype was due to a non-functional Pol ε, we created an allele called dpb2-201 that was identical to dpb2-200 except that the five codons after the Q687stop mutation were removed. This was done to minimize the risk of accidentally reading through the stop codon during translation. We first confirmed that the dpb2-201 allele was lethal in the low copy vector pRS314–dpb2-201 (Figure 1A). Next, we cloned the dpb2-201 allele into an over-expression vector under the control of the galactose-inducible promoter (plasmid pJL9-201) and co-expressed Dpb2-201 together with Pol2, Dpb3 and Dpb4. The Dpb2-201 protein was detected in a western blot of cell extracts obtained after induction with galactose (Figure 2A). Next, we applied a previously developed purification protocol for Pol ε that depends on ion-exchange chromatography and size-exclusion chromatography and found that Dpb2-201 did not co-purify with the other subunits (Figure 2B). This suggested that Dpb2-201 was unable to interact with the other Pol ε subunits.Figure 2.

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