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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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Holoenzyme assay with four-subunit Pol ε and the Pol2/Dpb3/Dpb4 complex. A single-primed pBluescript II SK(+) template was replicated by the indicated Pol ε complex in the presence of RPA and PCNA and in the presence (+) or absence (−) of RFC. Samples were collected after 1, 2, 4 and 8 min.
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gks880-F5: Holoenzyme assay with four-subunit Pol ε and the Pol2/Dpb3/Dpb4 complex. A single-primed pBluescript II SK(+) template was replicated by the indicated Pol ε complex in the presence of RPA and PCNA and in the presence (+) or absence (−) of RFC. Samples were collected after 1, 2, 4 and 8 min.

Mentions: We used three assays to explore the enzymatic properties of the purified Pol2/Dpb3/Dpb4 complex. The first assay measured the specific activity of the purified Pol2/Dpb3/Dpb4 complex with oligo(dT)-poly(dA) as the substrate. We found that the specific activity of the Pol2/Dpb3/Dpb4 complex was about 2-fold higher than that of the wild-type Pol ε (8860 U/mg and 4551 U/mg, respectively) suggesting that the catalytic activity of the complex was not negatively affected. Processivity is an important parameter for replicative polymerases, and previous studies had shown that Pol ε has a very high intrinsic processivity and that the two non-essential small subunits Dpb3 and Dpb4 are important for this processivity (24). Dpb2 was previously proposed to be located in a structural domain that interacted with the nascent double-stranded DNA and stabilized the interaction with the template (23). To address whether processivity is affected in the Pol2/Dpb3/Dpb4 complex, we performed the previously described experiments that had been performed with the Pol2/Dpb2 complex and wild-type Pol ε (24,40). The reaction conditions at an estimated 1:10 ratio of polymerase to primer were such that the measured termination probability at each position was constant from 2 to 8 min and <20% of the primer was extended. Thus, our experiment met the criteria for single-hit conditions (39). We found that the high processivity of Pol ε in the primer-extension assays was not affected when Dpb2 was missing from the Pol2/Dpb3/Dpb4 complex (Figure 4). There is, however, a strong pause site that limits the analysis to the first 64 nucleotides. This is still a substantial length in comparison to extension by Pol δ that has a processivity of only about five to seven nucleotides in the absence of the processivity clamp (40). Next, we asked whether the loss of Dpb2 affected the interaction of Pol ε with the processivity clamp, PCNA, that stimulates Pol ε in holoenzyme assays. We tested the functional interaction between Pol ε and PCNA on a single-primed circular single-stranded template and found that the loss of Dpb2 did not affect the PCNA-dependent stimulation (Figure 5). Thus, Dpb2 appears not to be essential for the interaction between Pol ε and double-stranded DNA, for achieving high processivity, for loading Pol ε onto the 3′-end of the primer, or for the interaction with PCNA.Figure 4.


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)

Holoenzyme assay with four-subunit Pol ε and the Pol2/Dpb3/Dpb4 complex. A single-primed pBluescript II SK(+) template was replicated by the indicated Pol ε complex in the presence of RPA and PCNA and in the presence (+) or absence (−) of RFC. Samples were collected after 1, 2, 4 and 8 min.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks880-F5: Holoenzyme assay with four-subunit Pol ε and the Pol2/Dpb3/Dpb4 complex. A single-primed pBluescript II SK(+) template was replicated by the indicated Pol ε complex in the presence of RPA and PCNA and in the presence (+) or absence (−) of RFC. Samples were collected after 1, 2, 4 and 8 min.
Mentions: We used three assays to explore the enzymatic properties of the purified Pol2/Dpb3/Dpb4 complex. The first assay measured the specific activity of the purified Pol2/Dpb3/Dpb4 complex with oligo(dT)-poly(dA) as the substrate. We found that the specific activity of the Pol2/Dpb3/Dpb4 complex was about 2-fold higher than that of the wild-type Pol ε (8860 U/mg and 4551 U/mg, respectively) suggesting that the catalytic activity of the complex was not negatively affected. Processivity is an important parameter for replicative polymerases, and previous studies had shown that Pol ε has a very high intrinsic processivity and that the two non-essential small subunits Dpb3 and Dpb4 are important for this processivity (24). Dpb2 was previously proposed to be located in a structural domain that interacted with the nascent double-stranded DNA and stabilized the interaction with the template (23). To address whether processivity is affected in the Pol2/Dpb3/Dpb4 complex, we performed the previously described experiments that had been performed with the Pol2/Dpb2 complex and wild-type Pol ε (24,40). The reaction conditions at an estimated 1:10 ratio of polymerase to primer were such that the measured termination probability at each position was constant from 2 to 8 min and <20% of the primer was extended. Thus, our experiment met the criteria for single-hit conditions (39). We found that the high processivity of Pol ε in the primer-extension assays was not affected when Dpb2 was missing from the Pol2/Dpb3/Dpb4 complex (Figure 4). There is, however, a strong pause site that limits the analysis to the first 64 nucleotides. This is still a substantial length in comparison to extension by Pol δ that has a processivity of only about five to seven nucleotides in the absence of the processivity clamp (40). Next, we asked whether the loss of Dpb2 affected the interaction of Pol ε with the processivity clamp, PCNA, that stimulates Pol ε in holoenzyme assays. We tested the functional interaction between Pol ε and PCNA on a single-primed circular single-stranded template and found that the loss of Dpb2 did not affect the PCNA-dependent stimulation (Figure 5). Thus, Dpb2 appears not to be essential for the interaction between Pol ε and double-stranded DNA, for achieving high processivity, for loading Pol ε onto the 3′-end of the primer, or for the interaction with PCNA.Figure 4.

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