ABSTRACT
The B-subunits of replicative DNA polymerases from Archaea to humans belong to the same protein family, suggesting that they share a common fundamental function. We report here the gene structure for the B-subunit of human DNA polymerase epsilon (POLE2), whose expression and transcriptional regulation is typical for replication proteins with some unique features. The 75 bp core promoter region, located within exon 1, contains an Sp1 element that is a critical determinant of promoter activity as shown by the luciferase reporter, electrophoretic mobility shift and DNase I footprinting assays. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Binding sites for E2F1 and NF-1 reside immediately downstream from the core promoter region. Our results suggest that human POLE2 is regulated by two E2F-pocket protein complexes, one associated with Sp1 and the other with NF-1. So far, only one replicative DNA polymerase B-subunit gene promoter, POLA2 encoding the B-subunit of DNA polymerase alpha, has been characterized. Mitogenic activation of the POLE2 promoter by an E2F-mediated mechanism resembles that of POLA2, but the regulation of basal promoter activity is different between these two genes.
Subject(s)
Carrier Proteins , Cell Cycle Proteins , DNA Polymerase II/genetics , Promoter Regions, Genetic/genetics , Response Elements/genetics , Sp1 Transcription Factor/metabolism , Transcription Factors/metabolism , Base Sequence , Cell Division , Culture Media, Serum-Free , DNA/genetics , DNA/metabolism , DNA Footprinting , DNA-Binding Proteins/metabolism , Deoxyribonuclease I/metabolism , E2F Transcription Factors , E2F1 Transcription Factor , Enzyme Induction , Exons/genetics , HeLa Cells , Humans , Introns/genetics , Macromolecular Substances , Models, Genetic , Molecular Sequence Data , Mutation/genetics , NFI Transcription Factors , Nuclease Protection Assays , Protein Binding , RNA, Messenger/analysis , RNA, Messenger/genetics , Retinoblastoma-Binding Protein 1 , Transcription, Genetic/geneticsABSTRACT
Topoisomerase IIbeta-binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharomyces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101. We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase epsilon. In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication. Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks. DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G(1) phase. We also show that TopBP1 interacts with the checkpoint protein hRad9. Thus, these results implicate TopBP1 in replication and checkpoint functions.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/physiology , DNA Damage , DNA Repair , DNA Replication , DNA-Binding Proteins , Drosophila Proteins , Saccharomyces cerevisiae Proteins , Schizosaccharomyces pombe Proteins , Transglutaminases , Animals , BRCA1 Protein/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cell Nucleus/metabolism , Chromosome Mapping , Cloning, Molecular , Cytoplasm/metabolism , DNA Polymerase II/metabolism , DNA, Complementary/metabolism , Drosophila , Ecdysone/metabolism , Fungal Proteins/chemistry , HeLa Cells , Humans , In Situ Hybridization, Fluorescence , Mice , Nuclear Proteins , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , S Phase , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/metabolism , Signal Transduction , Time Factors , Tumor Cells, Cultured , Two-Hybrid System TechniquesABSTRACT
In order to shed light on the role of mammalian DNA polymerase epsilon we studied the expression of mRNA for the human enzyme during cell proliferation and during the cell cycle. Steady-state levels of mRNA encoding DNA polymerase epsilon were elevated dramatically when quiescent (G0) cells were stimulated to proliferate (G1/S) in a similar manner to those of DNA polymerase alpha. Message levels of DNA polymerase beta were unchanged in similar experiments. The concentration of immunoreactive DNA polymerase epsilon was also much higher in extracts from proliferating tissues than in those from non-proliferating or slowly proliferating tissues. The level of DNA polymerase epsilon mRNA in actively cycling cells synchronized with nocodazole and in cells fractionated by counterflow centrifugal elutriation showed weaker variation, being at its highest at the G1/S stage boundary. The results presented strongly suggest that mammalian DNA polymerase epsilon is involved in the replication of chromosomal DNA and/or in a repair process that may be substantially activated during the replication of chromosomal DNA. A hypothetical role for DNA polymerase epsilon in a repair process coupled to replication is discussed.
