RESUMEN
Bloom's syndrome is a rare autosomal recessive genetic disorder characterized by chromosomal aberrations, genetic instability, and cancer predisposition, all of which may be the result of abnormal signal transduction during DNA damage recognition. Here, we show that BLM is an intermediate responder to stalled DNA replication forks. BLM colocalized and physically interacted with the DNA damage response proteins 53BP1 and H2AX. Although BLM facilitated physical interaction between p53 and 53BP1, 53BP1 was required for efficient accumulation of both BLM and p53 at the sites of stalled replication. The accumulation of BLM/53BP1 foci and the physical interaction between them was independent of gamma-H2AX. The active Chk1 kinase was essential for both the accurate focal colocalization of 53BP1 with BLM and the consequent stabilization of BLM. Once the ATR/Chk1- and 53BP1-mediated signal from replicational stress is received, BLM functions in multiple downstream repair processes, thereby fulfilling its role as a caretaker tumor suppressor.
Asunto(s)
Síndrome de Bloom/enzimología , ADN Helicasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas/metabolismo , Fase S , Western Blotting , Bromodesoxiuridina/metabolismo , Proteínas Portadoras , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , ADN Helicasas/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Hidroxiurea/farmacología , Cinética , Microscopía Confocal , Fosfoproteínas , Fosforilación , Pruebas de Precipitina , ARN Interferente Pequeño , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
When a double-strand break (DSB) forms in DNA, many molecules of histone H2AX present in the chromatin flanking the break site are rapidly phosphorylated. The phosphorylated derivative of H2AX is named gamma-H2AX, and the phosphorylation site is a conserved serine four residues from the C-terminus, 139 in mammals and 129 in budding yeast. An antibody to gamma-H2AX reveals that the molecules form a gamma-focus at the DSB site. The gamma-focus increases in size rapidly for 10-30 min after formation, and remains until the break is repaired. Studies have revealed that small numbers of gamma-foci are present in cells even without the purposeful introduction of DNA DSBs. These cryptogenic foci increase in number during senescence in culture and aging in mice. This chapter presents techniques for revealing gamma-H2AX foci in cultured cells, in metaphase spreads from cultured cells, in tissues, and in yeast.