Protein affinity chromatography reveals cell cycle dependent association of cellular factors with human DNA polymerase α

Chromosoma ◽  
1992 ◽  
Vol 102 (S1) ◽  
pp. S114-S120 ◽  
Author(s):  
Lars Rogge ◽  
Teresa S. -F. Wang
Keyword(s):  
Cell Cycle ◽  
Affinity Chromatography ◽  
Dna Polymerase ◽  
Dna Polymerase Α ◽  
Protein Affinity ◽  
Human Dna ◽  
Cellular Factors ◽  
Cycle Dependent

scholarly journals Molecular cloning of the cDNA for the catalytic subunit of plant DNA polymerase α and its cell-cycle dependent expression

1997 ◽  
Vol 2 (11) ◽  
pp. 695-709 ◽  
Author(s):  
Masayuki Yokoi ◽  
Masaki Ito ◽  
Masako Izumi ◽  
Hiroshi Miyazawa ◽  
Hirokazu Nakai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Cloning ◽  
Dna Polymerase ◽  
Catalytic Subunit ◽  
Dna Polymerase Α ◽  
Plant Dna ◽  
Cycle Dependent

scholarly journals Cell Cycle-Dependent Regulation of Human DNA Polymerase α-Primase Activity by Phosphorylation

1999 ◽  
Vol 19 (1) ◽  
pp. 646-656 ◽  
Author(s):  
Christian Voitenleitner ◽  
Christoph Rehfuess ◽  
Melissa Hilmes ◽  
Lynda O’Rear ◽  
Pao-Chi Liao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Polymerase ◽  
Cyclin E ◽  
Cyclin A ◽  
Human Cells ◽  
Dependent Manner ◽  
Dna Polymerase Α ◽  
A Cell ◽  
Cycle Dependent

ABSTRACT DNA polymerase α-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase α-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase α-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase α-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase α-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.

scholarly journals Cell cycle-dependent phosphorylation of human DNA polymerase alpha.

1991 ◽  
Vol 266 (12) ◽  
pp. 7893-7903
Author(s):  
H P Nasheuer ◽  
A Moore ◽  
A F Wahl ◽  
T S Wang
Keyword(s):  
Cell Cycle ◽  
Dna Polymerase ◽  
Dna Polymerase Alpha ◽  
Human Dna ◽  
Cycle Dependent

scholarly journals Regulation of human DNA polymerase delta during the cell cycle.

1994 ◽  
Vol 269 (39) ◽  
pp. 24027-24033
Author(s):  
X.R. Zeng ◽  
H. Hao ◽  
Y. Jiang ◽  
M.Y. Lee
Keyword(s):  
Cell Cycle ◽  
Dna Polymerase ◽  
Dna Polymerase Delta ◽  
Human Dna

scholarly journals Mutational Studies of Human DNA Polymerase α

1995 ◽  
Vol 270 (37) ◽  
pp. 21563-21570 ◽  
Author(s):  
Qun Dong ◽  
Teresa S.-F. Wang
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerase Α ◽  
Human Dna

Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle

1988 ◽  
Vol 8 (11) ◽  
pp. 5016-5025
Author(s):  
A F Wahl ◽  
A M Geis ◽  
B H Spain ◽  
S W Wong ◽  
D Korn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Steady State ◽  
Dna Polymerase ◽  
Transformed Cells ◽  
Mrna Levels ◽  
Dna Polymerase Alpha ◽  
Human Dna ◽  
Alpha Gene

We studied the expression of the human DNA polymerase alpha gene during cell proliferation, during cell progression through the cell cycle, and in transformed cells compared with normal cells. During the activation of quiescent cells (G0 phase) to proliferate (G1/S phases), the steady-state mRNA levels, rate of synthesis of nascent polymerase protein, and enzymatic activity in vitro exhibited a substantial and concordant increase prior to the peak of in vivo DNA synthesis. In transformed cells, the respective values were amplified greater than 10-fold. In actively growing cells separated into discrete stages of the cell cycle by counterflow elutriation or by mitotic shakeoff, levels of steady-state transcripts, translation rates, and enzymatic activities of polymerase alpha were constitutively and concordantly expressed at all stages of the cell cycle, with only a moderate elevation prior to the S phase and a slight decline in the G2 phase. These findings support the conclusion that the regulation of human DNA polymerase alpha gene expression is at the transcriptional level and strongly suggest that the regulatory mechanisms that are operative during the entrance of a cell into the mitotic cycle are fundamentally different from those that modulate polymerase alpha expression in continuously cycling cells.

scholarly journals p110 CUX1 Cooperates with E2F Transcription Factors in the Transcriptional Activation of Cell Cycle-Regulated Genes

2008 ◽  
Vol 28 (10) ◽  
pp. 3127-3138 ◽  
Author(s):  
Mary Truscott ◽  
Ryoko Harada ◽  
Charles Vadnais ◽  
François Robert ◽  
Alain Nepveu
Keyword(s):  
Cell Cycle ◽  
Dna Polymerase ◽  
Transcriptional Activation ◽  
Affinity Purification ◽  
Gene Promoter ◽  
Dominant Negative ◽  
In Vitro Assays ◽  
Dominant Negative Mutant ◽  
Dna Polymerase Α ◽  
Reporter Assays

ABSTRACT The transcription factor p110 CUX1 was shown to stimulate cell proliferation by accelerating entry into S phase. As p110 CUX1 can function as a transcriptional repressor or activator depending on promoter context, we investigated its mechanism of transcriptional activation using the DNA polymerase α gene promoter as a model system. Linker-scanning analysis revealed that a low-affinity E2F binding site is required for transcriptional activation. Moreover, coexpression with a dominant-negative mutant of DP-1 suggested that endogenous E2F factors are indeed needed for p110-mediated activation. Tandem affinity purification, coimmunoprecipitation, chromatin immunoprecipitation, and reporter assays indicated that p110 CUX1 can engage in weak protein-protein interactions with E2F1 and E2F2, stimulate their recruitment to the DNA polymerase α gene promoter, and cooperate with these factors in transcriptional activation. On the other hand, in vitro assays suggested that the interaction between CUX1 and E2F1 either is not direct or is regulated by posttranslational modifications. Genome-wide location analysis revealed that targets common to p110 CUX1 and E2F1 included many genes involved in cell cycle, DNA replication, and DNA repair. Comparison of the degree of enrichment for various E2F factors suggested that binding of p110 CUX1 to a promoter will favor the specific recruitment of E2F1, and to a lesser extent E2F2, over E2F3 and E2F4. Reporter assays on a subset of common targets confirmed that p110 CUX1 and E2F1 cooperate in their transcriptional activation. Overall, our results show that p110 CUX1 and E2F1 cooperate in the regulation of many cell cycle genes.

scholarly journals Human DNA polymerase α interacts with mismatch repair proteins MSH2 and MSH6

FEBS Letters ◽  
2016 ◽  
Vol 590 (23) ◽  
pp. 4233-4241 ◽  
Author(s):  
Harri M. Itkonen ◽  
Jukka Kantelinen ◽  
Markku Vaara ◽  
Sinikka Parkkinen ◽  
Bernhard Schlott ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Dna Polymerase ◽  
Dna Polymerase Α ◽  
Human Dna ◽  
Mismatch Repair Proteins
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