Immunoaffinity purification of DNA polymerase δ1

Jiang, Yunquan; Zhang, Shan-Jian; Wu, Shuang‐Qing; Lee, Marietta Y.W.T.

doi:10.1016/0003-9861(95)90013-6

Cited by 46 publications

(31 citation statements)

References 35 publications

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“…Towards this goal, in vitro pull-down assays were performed using bacterially expressed full-length GSTpRb (residues 1 ± 928) using lysates from the breast epithelial cell line, MCF10A, and the breast cancer cell line, MCF7. After puri®cation on glutathione sepharose, GST-pRb interacting proteins were eluted with excess glutathione and analysed by Western blotting using monoclonal (78F5) antibodies that recognize p125 (Jiang et al, 1995). As shown in Figure 1, p125 found in MCF10A and MCF7 cell lysates was able to bind to GST-pRb (lanes 3 and 6), but not to GST alone (lanes 2 and 5).…”

Section: Resultsmentioning

confidence: 99%

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Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase δ (p125)

et al. 2000

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The retinoblastoma gene product (pRb) interacts with many cellular proteins to function in the control of cell division, di erentiation, and apoptosis. Several pRb binding proteins complex with pRb through an amino acid sequence called the LXCXE motif. The catalytic subunit of DNA polymerase d (p125) contains a LXCXE motif. To further study the biochemical function of this polymerase, we sought to determine if p125 interacts with pRb. Experiments using GST-pRb fusion proteins showed that p125 from breast epithelial (MCF10A) cell extracts associates with pRb. In addition, GST-p125 fusion proteins bound pRb from the same cell extracts. The pRb that associated with GST-p125 was largely unphosphorylated. Coimmunoprecipitation experiments using cell cycle synchronized cells revealed that p125 and pRb form a complex predominantly during G 1 phase, the phase during which pRb is mostly unphosphorylated. In vitro phosphorylation of GST-pRb by the cyclin dependent kinases reduced the ability of p125 to associate with GST-pRb. Addition of the LXCXE containing protein SV40 large T antigen to GST-pRb blocks the ability of p125 to associate with pRb, suggesting that it may be through a LXCXE sequence by which p125 interacts with pRb. Finally, in vitro polymerase assays demonstrate that GST-pRb fusion protein stimulates DNA polymerase d activity.

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Section: Resultsmentioning

confidence: 99%

“…DNA polymerase d was puri®ed from calf thymus using an immunoa nity column (Jiang et al, 1995) and polymerase activity was measured in the presence of nanogram quantities of GST or GST-pRb fusion proteins. As shown in Figure 7, GST-pRb increased the activity of puri®ed DNA polymerase d 2 ± 3-fold relative to GST controls.…”

Section: Resultsmentioning

confidence: 99%

Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase δ (p125)

et al. 2000

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“…Calf thymus DNA pol δ was purified as described previously. 31 The peak fractions from the immunoaffinity column were used for histone kinase assays. Kinase assays were performed in a final volume of 30 µl containing 1 µg Histone H1 (Sigma) in 1 x histonekinase buffer.…”

Section: Methodsmentioning

confidence: 99%

“…4 The CDK2/cylcinA complex is associated with the pol δ complex both from HeLa cells and calf thymus after anti-p125 immunoaffinity column chromatography (data not shown). Calf thymus DNA pol δ was purified using an anti-p125 immunoaffinity column, 31 and the eluted fractions were assayed for pol δ activity using poly(dA)/oligo(dT) as the template (Fig. 4A, circles) as well as for histone kinase activity (Fig.…”

Section: A B C Dmentioning

confidence: 99%

“…(A) Association of a histone kinase activity with highly purified calf thymus pol δ. Calf thymus DNA pol δ was purified as described previously. 31 The fractions of pol δ from the anti-p125 immunoaffinity column were assayed for pol δ activity. The peak fractions were also incubated with histone H1 for histone kinase activity in the presence of [γ-32 P]ATP.…”

Section: A B C Dmentioning

confidence: 99%

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Direct Interaction of p21 with p50, the Small Subunit of Human DNA Polymerase Delta

Xie²,

Rahmeh³

et al. 2006

Cell Cycle

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2425 KEY WORDS ReportDirect Interaction of p21 with p50, the Small Subunit of Human DNA Polymerase Delta ABSTRACT Using a yeast two-hybrid screening technique and the p50 subunit of human DNA polymerase delta (pol δ) as a bait, p21 was found to interact with the p50 subunit of pol δ. A direct interaction between p21 and p50 was confirmed by using ELISA and pull-down assays with purified proteins. The interaction sites between p50 and p21 were mapped by pull down assays with GST deletion mutants. Residues 127-193 constitute the primary interaction region on p50 to which p21 binds, while p50 binds to the C-terminal 26 residues of p21. A histone kinase activity was associated with the highly purified calf thymus pol δ and addition of purified recombinant p21 inhibited the kinase activity in a dose dependent manner. p50 is phosphorylated in vivo and can be phosphorylated by CDK2/cyclinA in vitro. In vivo evidence of p21 association with p50 was obtained by coimmunoprecipitation using MCF7 cells. It was also shown that the association of p21 with p50 and other components of the pol δ complex increased in MCF7 cells treated with adriamycin. Our results suggested that p50 might target or anchor p21 to pol δ complex upon certain DNA damage such as adriamycin treatment.

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Purification of Protein Complexes by Immunoaffinity Chromatography: Application to Transcription Machinery

Thompson

Jensen

Lamberski

et al.

Genetic Engineering: Principles and Methods

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To be useful for immunoaffinity chromatography, an antibody must have high affinity for its antigen in order to bind the antigen in a dilute solution. This property, however, generally results in a complex that is difficult to disrupt in order to recover the antigen in an active form. Most elution conditions require extremes of pH values, chaotropic salts, or denaturing reagents. A unique type of mAb has high affinity for a protein antigen, but whose interaction with the antigen can be dissociated under gentle elution conditions (7,8). This type of mAb has been designated a polyol-responsive mAb (PR-mAb) because the eluting buffer is a neutral, aqueous buffer containing a low molecular weight polyhydroxylated compound (polyol) and a nonchaotropic salt, combinations that are 82 N. THOMPSON, ET AL.

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Immunoaffinity purification of DNA polymerase δ1

Cited by 46 publications

References 35 publications

Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase δ (p125)

Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase δ (p125)

Direct Interaction of p21 with p50, the Small Subunit of Human DNA Polymerase Delta

Purification of Protein Complexes by Immunoaffinity Chromatography: Application to Transcription Machinery

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