Control of DNA replication in vivo: study of mouse models for Cdc6 and Cdt1 overexpression

• Autores: Sabela Bua Aguín
• Directores de la Tesis: Juan Méndez Zunzunegui (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2013
• Idioma: inglés
• Tribunal Calificador de la Tesis: Luis Blanco Dávila (presid.), Óscar Fernández Capetillo (secret.), Raimundo Freire Betancor (voc.), María Gómez Vicentefranqueira (voc.), David Santamaría Velilla (voc.)
• Materias:
  • Química
    • Bioquímica
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • DNA! replication! is! a! biological! process! that! leads! to! the! transmission! of! genetic! information!to!the!next!cell!generation.!Failure!to!maintain!a!tight!control!on!DNA!replication! may!result!in!genome!underreplication!or!overreplication,!which!have!been!linked!to!a!variety! of!human!diseases,!including!cancer.!Origins!are!licensed!for!replication!from!late!mitosis!to!G1! by! the! coordinated! action! of! the! Origin! Recognition! Complex! (ORC),! Cdc6! and! Cdt1,! which! promote!the!loading!of!the!MCM2Z7!helicase!onto!DNA.!Previous!studies!in!yeast,"human!and! mouse!cells!have!shown!that!insufficient!origin!licensing!through!MCM!downregulation!leads! to! genomic! instability! and! oncogenesis!in" vivo.! Nevertheless! much! less! is! known! about! the! effects! of! overexpressing! `licensing! factors¿! such! as! Cdc6! and! Cdt1! in" vivo,! although! both! factors! have! been! reported! to! have! oncogenic!activity! in! culture.!In! order! to! characterize!in" vivo!the!effects!of!Cdc6!and!Cdt1!deregulated!expression!we!have!generated!inducible!mouse! models! for! Cdc6! and! Cdt1!proteins.! High! Cdc6! levels! enhance! MCM! chromatin! association! both!in" vivo! and!in" vitro.! In! contrast,! Cdt1! overexpression! did! not! increase!MCM! chromatin! loading,! suggesting! that! Cdc6! is! the! limiting! factor! for!this! reaction.! At! the! molecular! level,! Cdc6!overexpression!changes!the!dynamics!of!DNA!replication!by!increasing!the!frequency!of! origin!firing!and!decreasing!the!fork!progression!rate!to!an!extent!that!cannot!be!significantly! enhanced! under! conditions! of! replicative! stress,! suggesting! that! upon! Cdc6! overexpression! most!of!the!potential!origins!are!fired.!Although!no!defects!on!cell!proliferation!were!observed! in!shortZterm!cultured!cells,!TetONZCDC6!mice!are!prone!to!developing!histiocytic!sarcomas!and! BZcell!lymphomas!and!have!a! shorter!lifespan.!In!contrast,!a! tissueZspecific! transgenic!model! generated! in! parallel,! K5ZCDC6tg,! showed! a! normal! lifespan! but!mice! were! hypersensitive! to! chemicallyZinduced! skin! tumorigenesis.! Interestingly,! K5ZCDC6tg!mice! display! a! skinZspecific! antiZageing!phenotype!that!might!be!related!to!a!delayed!progression!of!the!hair!growth!cycle.! When!Cdc6!and!Cdt1!overexpression!were!combined!by!crossbreeding!TetONZCDC6/CDT1!mice,! MEFs!undergo!partial!DNA!reZreplication,!activating!the!DNA!damage!response!and!apoptotic! programs.!Strikingly,!mice!overexpressing!Cdc6!and!Cdt1!displayed!morbidity!signs!in!few!days.! Proliferative!tissues!showed!severe!cytoarchitectural!abnormalities,!increased!mitotic!activity,! DNA!damage!response!activation!and!increased!apoptosis.!This!Thesis!provides!evidence!that! aberrant! expression! of! Cdc6! and! Cdc6/Cdt1! threatens! genomic! stability! and! promotes! cell! transformation!in"vivo.!