Studies on origin binding proteins involved in mammalian DNA replication

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, BAlKu 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) CH 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 CH 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, 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 RSUM L'objectif de la recherche de cette thse est d'examiner les interactions entre les proteines et les squences de l'ADN qui contiennent des origines de rplication in vivo.Auparavant, nous avons identif deux protines, BA/K.uet CBP/1433, capables de se lier aux origines de rplication.Nous avons aussi dmontr que Ku intragit in vitro avec une squence d'origine de 36-pb, A3/4.Les protines 14-3-3 ont aussi t identifies pralablement d'tre capable de se lier aux structures cruciformes in vitro qui se retrouvent dans les origines de rplication de l'ADN.Les interactions entre Ku et 14-3-3 avec les origines mammifres ors8 et ors12 ont t analyses par le traitement des cellules de singe (CV-l) avec du formaldehyde suivi par l'immunoprcipitation de la chromatine (IPCh) et la quantification PCR de l'ADN.Un essai in vitro pour analyser le rle de 14-3-3 dans la replication de l'ADN a t utilis, en employant des anticorps contre les isoformes 14-3-3[3, , y, et (J.Ku et les isoformes 14-3-3 [3, , y, et (J ont t identifis in vivo aux origines de rplication et l'association de ces proteines avec ces origines est maximale la phase G liS.La rplication de p 186, un plasmid contenant une origine de rplication, a t inhibe par l'addition des anticorps anti-14-3-3[3, , y, ou (J de -30-70%.Les lignes cellulaires mutantes (xrs-5) ou dficientes en Ku80 (Ku80-1 -) ont aussi t testes pour leur capacit repliquer p186.Les extraits cytoplasmiques et totaux de xrs-5 avaient une activit de rplication semblable aux cellules CH avec le phnotype sauvage, mais les extraits nuclaires de xrs-5 ou les extraits totaux des cellules dficientes Ku80-1 -ne possdaient pas d'activit de rplication, ou une rplication trs rduite de -70%.L'association de Ku avec les origines de rplication DHFR ori[3 ou ADA prsentes dans les cellules xrs-5 ou Ku80-1 -, a aussi t teste in vivo.Les anticorps anti-Ku n'ont pas immunoprecipit un montant dtectable de Ku dans les cellules xrs-5 ou Ku80-1 -, contrairement aux cellules de phnotype sauvage o Ku a t dtect aux origines de rplication DHFR ori[3 et ADA.Ces rsultats impliquent Ku dans la rplication de l'ADN et suggmte l' xistence d'mle autre protine capable de remplir le rle de Ku.Ils indiquent aussi un nouveau role pour Ku et les 14-3-3 e, [3, y, et (J isoformes dans la rplication de l'ADN mammifre, et v permettent une meilleure comprhension de l'association chromosomique des protines impliques dans la rplication, avec les squences de l'ADN qui contiennent des origines de rplication mammifre.

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