Studies on origin binding proteins involved in mammalian DNA replication
Bibliographic record
Abstract
The objective of this thesis is to investigate the proteins interacting with specific DNA sequences, termed origins of DNA replication in vivo.Two previously described origin binding proteins, ûBAlKu and CBPI14-3-3 were analyzed.Previously, Ku was shown to bind to A3/4, a 36-bp origin sequence, in vitro, and 14-3-3 isoforms were identified as cruc:iform binding proteins (CBP) which interact with cruciform structures present in mammalian replication origins.Here, the in vivo association of Ku and 14-3-3 with mammalian origins of DNA replication was analyzed by studying its association with the monkey (CV-l) replication origins ors 8 and ors12, by formaldehyde cross-linking, followed by chromatin immunoprecipitation (Chip) and quantitative PCR analysis.The involvement of 14-3-3 in mammalian DNA replication was also analyzed by studying the effect of anti-14-3-313, E, y, and c.; antibodies in the in vitro replication of p186, a plasmid containing the minimal replication origin of ors8.Ku and 14-3-3 13, E, y, c.; and 0' isoforms were found to be associated with mammalian origins of DNA replication and their association was the highest in cells synchronized at the GUS phase of the cell cycle.In addition, Anti-14-3-3E, y and c.; antibodies inhibited p186 replication by approximately 30-80%.The Ku80 mutant (xrs-5) and deficient (Ku80-1 -MEFs) celllines were also tested for their ability to replicate p186, in vitro.Whole cell (WCB) and cytoplasmic cell extracts from the xrs-5 cells replicated p186 with the same efficiency as are wild-type (wt) CHû KI cells.In contrast, xrs-5 nuclear extracts did not possess any detectable replication activity, while the Ku80-1 -WCB had a decrease of ~70% in their ability to support p186 replication, by comparison to the wt Ku80+ 1 + extracts.Furthermore, in vivo, the p186 episomal DNA replication in transfected xrs-5 cells was reduced by 45% by comparison to CHû KI cells.The in vivo association of Ku with the Chinese hamster DHFR oril3 or the mou se Adenosine deaminase (ADA) origins of DNA replication was examined in both the Ku80 mutant (xrs-S) and deficient (Ku80-/ ) cell1ines, and in their respective wild-type counterparts.Anti-Ku antibodies failed to immunoprecipitate a detectable amount of Ku from the either xrs-5 or Ku80-1 -cells in the origin-containing-sequence, in contrast to the wild type cells, wherein Ku was found to be associated with the oril3 and ADA origins, respectively. IIIThe data implicate Ku antigen in DNA replication and suggest the existence of another protein in rodent cells that is able to substitute for Ku function.They also indicate a novel function for Ku and the 14-3-3 isoforms p, E, y, ç and Ci, as origin-binding-proteins in vivo, which provides a better understanding of the chromosomal association and DNA binding sequences of mammalian initiator proteins with origins of replication in their natural chromosomal environment.IV RÉSUMÉ L'objectif de la recherche de cette thèse est d'examiner les interactions entre les proteines et les séquences de l'ADN qui contiennent des origines de réplication in vivo.Auparavant, nous avons identifé deux protéines, ûBA/K.uet CBP/1433, capables de se lier aux origines de réplication.Nous avons aussi démontré que Ku intéragit in vitro avec une séquence d'origine de 36-pb, A3/4.Les protéines 14-3-3 ont aussi été identifiées préalablement d'être capable de se lier aux structures cruciformes in vitro qui se retrouvent dans les origines de réplication de l'ADN.Les interactions entre Ku et 14-3-3 avec les origines mammifères ors8 et ors12 ont été analysées par le traitement des cellules de singe (CV-l) avec du formaldehyde suivi par l'immunoprécipitation de la chromatine (IPCh) et la quantification PCR de l'ADN.Un essai in vitro pour analyser le rôle de 14-3-3 dans la replication de l'ADN a été utilisé, en employant des anticorps contre les isoformes 14-3-3[3, ç, y, ç et (J.Ku et les isoformes 14-3-3 [3, ç, y, ç et (J ont été identifiés in vivo aux origines de réplication et l'association de ces proteines avec ces origines est maximale à la phase G liS.La réplication de p 186, un plasmid contenant une origine de réplication, a été inhibée par l'addition des anticorps anti-14-3-3[3, ç, y, ç ou (J de -30-70%.Les lignées cellulaires mutantes (xrs-5) ou déficientes en Ku80 (Ku80-1 -) ont aussi été testées pour leur capacité à repliquer p186.Les extraits cytoplasmiques et totaux de xrs-5 avaient une activité de réplication semblable aux cellules CHû avec le phénotype sauvage, mais les extraits nucléaires de xrs-5 ou les extraits totaux des cellules déficientes Ku80-1 -ne possédaient pas d'activité de réplication, ou une réplication très réduite de -70%.L'association de Ku avec les origines de réplication DHFR ori[3 ou ADA présentes dans les cellules xrs-5 ou Ku80-1 -, a aussi été testée in vivo.Les anticorps anti-Ku n'ont pas immunoprecipité un montant détectable de Ku dans les cellules xrs-5 ou Ku80-1 -, contrairement aux cellules de phénotype sauvage où Ku a été détecté aux origines de réplication DHFR ori[3 et ADA.Ces résultats impliquent Ku dans la réplication de l'ADN et suggèmte l' éxistence d'mle autre protéine capable de remplir le rôle de Ku.Ils indiquent aussi un nouveau role pour Ku et les 14-3-3 e, [3, y, ç et (J isoformes dans la réplication de l'ADN mammifère, et v permettent une meilleure compréhension de l'association chromosomique des protéines impliquées dans la réplication, avec les séquences de l'ADN qui contiennent des origines de réplication mammifère.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".