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Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation
Using libraries of replication origins generated previously, we identified three clones that supported the autonomous replication of their respective plasmids in transformed, but not in normal cells. Assessment of their in vivo replication activity by in situ chromosomal DNA replication assays revea...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2010
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2853114/ https://www.ncbi.nlm.nih.gov/pubmed/20064876 http://dx.doi.org/10.1093/nar/gkp1192 |
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author | Di Paola, Domenic Rampakakis, Emmanouil Chan, Man Kid Arvanitis, Dina N. Zannis-Hadjopoulos, Maria |
author_facet | Di Paola, Domenic Rampakakis, Emmanouil Chan, Man Kid Arvanitis, Dina N. Zannis-Hadjopoulos, Maria |
author_sort | Di Paola, Domenic |
collection | PubMed |
description | Using libraries of replication origins generated previously, we identified three clones that supported the autonomous replication of their respective plasmids in transformed, but not in normal cells. Assessment of their in vivo replication activity by in situ chromosomal DNA replication assays revealed that the chromosomal loci corresponding to these clones coincided with chromosomal replication origins in all cell lines, which were more active by 2–3-fold in the transformed by comparison to the normal cells. Evaluation of pre-replication complex (pre-RC) protein abundance at these origins in transformed and normal cells by chromatin immunoprecipitation assays, using anti-ORC2, -cdc6 and -cdt1 antibodies, showed that they were bound by these pre-RC proteins in all cell lines, but a 2–3-fold higher abundance was observed in the transformed by comparison to the normal cells. Electrophoretic mobility shift assays (EMSAs) performed on the most efficiently replicating clone, using nuclear extracts from the transformed and normal cells, revealed the presence of a DNA replication complex in transformed cells, which was barely detectable in normal cells. Subsequent supershift EMSAs suggested the presence of transformation-specific complexes. Mass spectrometric analysis of these complexes revealed potential new protein players involved in DNA replication that appear to correlate with cellular transformation. |
format | Text |
id | pubmed-2853114 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-28531142010-04-12 Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation Di Paola, Domenic Rampakakis, Emmanouil Chan, Man Kid Arvanitis, Dina N. Zannis-Hadjopoulos, Maria Nucleic Acids Res Genome Integrity, Repair and Replication Using libraries of replication origins generated previously, we identified three clones that supported the autonomous replication of their respective plasmids in transformed, but not in normal cells. Assessment of their in vivo replication activity by in situ chromosomal DNA replication assays revealed that the chromosomal loci corresponding to these clones coincided with chromosomal replication origins in all cell lines, which were more active by 2–3-fold in the transformed by comparison to the normal cells. Evaluation of pre-replication complex (pre-RC) protein abundance at these origins in transformed and normal cells by chromatin immunoprecipitation assays, using anti-ORC2, -cdc6 and -cdt1 antibodies, showed that they were bound by these pre-RC proteins in all cell lines, but a 2–3-fold higher abundance was observed in the transformed by comparison to the normal cells. Electrophoretic mobility shift assays (EMSAs) performed on the most efficiently replicating clone, using nuclear extracts from the transformed and normal cells, revealed the presence of a DNA replication complex in transformed cells, which was barely detectable in normal cells. Subsequent supershift EMSAs suggested the presence of transformation-specific complexes. Mass spectrometric analysis of these complexes revealed potential new protein players involved in DNA replication that appear to correlate with cellular transformation. Oxford University Press 2010-04 2010-01-11 /pmc/articles/PMC2853114/ /pubmed/20064876 http://dx.doi.org/10.1093/nar/gkp1192 Text en © The Author(s) 2010. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/2.5 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Genome Integrity, Repair and Replication Di Paola, Domenic Rampakakis, Emmanouil Chan, Man Kid Arvanitis, Dina N. Zannis-Hadjopoulos, Maria Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation |
title | Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation |
title_full | Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation |
title_fullStr | Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation |
title_full_unstemmed | Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation |
title_short | Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation |
title_sort | increased origin activity in transformed versus normal cells: identification of novel protein players involved in dna replication and cellular transformation |
topic | Genome Integrity, Repair and Replication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2853114/ https://www.ncbi.nlm.nih.gov/pubmed/20064876 http://dx.doi.org/10.1093/nar/gkp1192 |
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