Mechanism of Chromatin Replication

Descripción del proyecto

Nuevos conocimientos sobre la coordinación de procesos durante la duplicación cromosómica

La división de las células humanas implica la duplicación precisa de cerca de seis mil millones de pares de bases de ADN genómico, así como de todas las proteínas asociadas. La cromatina se empaqueta formando nucleosomas, que contienen ADN enrollado alrededor de un octámero de proteínas histonas, que se abren cada vez que se produce la replicación. Las modificaciones de histonas se restablecen en las hebras hijas justo después de la horquilla de replicación. La transmisión hereditaria de las histonas parentales se coordina con la deposición de nuevas histonas en los nucleosomas; y la heterocromatina represiva debe replicarse y restablecerse inmediatamente tras la replicación. El objetivo del proyecto MeChroRep, financiado con fondos europeos, es utilizar la reconstitución «in vitro» de la replicación de la cromatina, así como técnicas de genética molecular y biología estructural, a fin de mejorar la comprensión de la coordinación de la replicación del ADN durante el desensamblaje y reensamblaje de la cromatina, que resulta en una duplicación cromosómica precisa.

Objetivo

Each time a human cell divides, it must make an exact copy of its 46 chromosomes. This requires precise duplication of ~6 billion base pairs of genomic DNA and all its associated proteins. Mistakes in this process can lead to developmental defects and cancer. Chromatin is packaged as nucleosomes comprising 147bp of DNA wrapped around a core of eight histone proteins. The nucleosome is a stable structure which must be disrupted each time the double helix is accessed during replication. ‘Epigenetic’ information in the form of covalent histone modifications must be re-established on both daughter strands immediately behind the replication fork. Inheritance of parental histones with their covalent marks must be coordinated with deposition of new histones into nucleosomes. And repressive heterochromatin must be replicated and immediately re-established after replication.
Our goal is to achieve a deep understanding of how DNA replication is coordinated with the disassembly and reassembly of chromatin to ensure accurate chromosome duplication.
We will use in vitro reconstitution of chromatin replication along with molecular genetics and structural biology to:
• Generate a molecular view of how the budding yeast replication machinery, with other key factors, disrupts parental nucleosomes during replication.
• Determine how parental nucleosomes are transferred to the nascent daughter strands.
• Understand how deposition of new histones is coordinated with the inheritance of parental histones.
• Characterise how budding yeast heterochromatin is disrupted and re-established during replication.
• Begin studying this process using human proteins, focussing on areas where human replication mechanisms diverge from yeast.
This work will underpin our long-term goal of reconstituting functional chromosomes from defined components to understand how DNA replication interacts with gene expression, DNA repair and chromosome segregation.