Limits...
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.

Show MeSH

Related in: MedlinePlus

Pull-down experiment with GST-Pol2, GST-Dpb2 and GST–Dpb2-201. The positions of each subunit of Pol ε are indicated to the left. The amount of protein that was loaded in each lane was titrated such that an equal amount of GST-Pol2 was loaded in lanes 1 and 2, and equal amounts of GST-Dpb2 and GST–Dpb2-201 were loaded in lanes 3 and 4. The gel was colloidal Coomassie-stained (16).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks880-F3: Pull-down experiment with GST-Pol2, GST-Dpb2 and GST–Dpb2-201. The positions of each subunit of Pol ε are indicated to the left. The amount of protein that was loaded in each lane was titrated such that an equal amount of GST-Pol2 was loaded in lanes 1 and 2, and equal amounts of GST-Dpb2 and GST–Dpb2-201 were loaded in lanes 3 and 4. The gel was colloidal Coomassie-stained (16).

Mentions: To further test this hypothesis, we positioned a GST tag at the N-terminus of Pol2 in the over-expression system described above and asked whether over-expressed Dpb2 or Dpb2-201 would co-purify with GST-Pol2 on a glutathione-sepharose column. Only Dpb2, Dpb3 and Dpb4 co-purified with Pol ε over the affinity column (Figure 3, lanes 1 and 2), supporting our previous result that suggested that Dpb2-201 might have lost the ability to interact with Pol2 (Figure 2B). However, the western blots suggested that the Dpb2-201 protein was less abundant than wild-type Dpb2 when over-expressed in yeast (Figure 2A). This could potentially lead to a substoichiometric amount of Dpb2-201 in our Pol ε preparations and only give the impression that Dpb2-201 had lost its ability to interact with Pol2. Thus, we positioned a GST tag at the N-terminus of Dpb2 and Dpb2-201 to determine whether they would co-purify with Pol2 on a glutathione-sepharose column. Equal amounts of GST-Dpb2 and GST–Dpb2-201 were loaded in each lane. The amount of Pol2 co-purifying with GST–Dpb2-201 was significantly less compared with the GST-Dpb2 purification (Figure 3, lanes 3 and 4). Altogether, these results support a model in which Dpb2 depends on its C-terminus to interact with Pol2, but the small amount of Pol2 co-purifying with GST–Dpb2-201 suggests that there may exist a second, weak interaction surface. Furthermore, we observed a slight shift in the stoichiometry of Dpb3 and Dpb4 in the absence or presence of Dpb2 or GST-Dpb2.Figure 3.


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)

Pull-down experiment with GST-Pol2, GST-Dpb2 and GST–Dpb2-201. The positions of each subunit of Pol ε are indicated to the left. The amount of protein that was loaded in each lane was titrated such that an equal amount of GST-Pol2 was loaded in lanes 1 and 2, and equal amounts of GST-Dpb2 and GST–Dpb2-201 were loaded in lanes 3 and 4. The gel was colloidal Coomassie-stained (16).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks880-F3: Pull-down experiment with GST-Pol2, GST-Dpb2 and GST–Dpb2-201. The positions of each subunit of Pol ε are indicated to the left. The amount of protein that was loaded in each lane was titrated such that an equal amount of GST-Pol2 was loaded in lanes 1 and 2, and equal amounts of GST-Dpb2 and GST–Dpb2-201 were loaded in lanes 3 and 4. The gel was colloidal Coomassie-stained (16).
Mentions: To further test this hypothesis, we positioned a GST tag at the N-terminus of Pol2 in the over-expression system described above and asked whether over-expressed Dpb2 or Dpb2-201 would co-purify with GST-Pol2 on a glutathione-sepharose column. Only Dpb2, Dpb3 and Dpb4 co-purified with Pol ε over the affinity column (Figure 3, lanes 1 and 2), supporting our previous result that suggested that Dpb2-201 might have lost the ability to interact with Pol2 (Figure 2B). However, the western blots suggested that the Dpb2-201 protein was less abundant than wild-type Dpb2 when over-expressed in yeast (Figure 2A). This could potentially lead to a substoichiometric amount of Dpb2-201 in our Pol ε preparations and only give the impression that Dpb2-201 had lost its ability to interact with Pol2. Thus, we positioned a GST tag at the N-terminus of Dpb2 and Dpb2-201 to determine whether they would co-purify with Pol2 on a glutathione-sepharose column. Equal amounts of GST-Dpb2 and GST–Dpb2-201 were loaded in each lane. The amount of Pol2 co-purifying with GST–Dpb2-201 was significantly less compared with the GST-Dpb2 purification (Figure 3, lanes 3 and 4). Altogether, these results support a model in which Dpb2 depends on its C-terminus to interact with Pol2, but the small amount of Pol2 co-purifying with GST–Dpb2-201 suggests that there may exist a second, weak interaction surface. Furthermore, we observed a slight shift in the stoichiometry of Dpb3 and Dpb4 in the absence or presence of Dpb2 or GST-Dpb2.Figure 3.

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