Eukaryotic DNA replication: a single-molecule approach to the study of yeast replication on chromatin

Objetivo

DNA replication is essential to cellular function. During a lifetime, each of us synthesizes a light-year’s length of DNA, but this process is so robust that few of us will develop cancer. In eukaryotes, DNA is packed into chromatin, a hierarchical DNA-protein assembly of which the nucleosome forms the basic unit. Chromatin replication convolves DNA replication with the duplication and reassembly of all DNA-associated proteins. Understanding the coupling between these processes has fundamental implications for epigenetic inheritance and cancer.
The goal of this proposal is to gain spatiotemporal insight into chromatin replication by using our biophysical expertise in replication and chromosomal dynamics to build up a mechanistic timeline of the process. We will harness recent advances in the reconstitution of the yeast replisome alongside our novel, high-throughput single-molecule approach to visualize and quantify the collaboration between a single yeast replisome and the histone chaperones to achieve chromatin replication. We will:
•Monitor the assembly of the replisome on chromatin and visualize how nucleosomes impact its progression.
•Quantify how the replisome and histone chaperones disrupt nucleosomes and retain histones for further processing.
•Detect the deposition of newly synthesized histones behind the replisome and reveal the interactions between replisome components and histone chaperones that couple replication to nucleosome assembly.
•Report on the phenomenon of epigenetic inheritance by imaging histone recycling between parental and daughter DNA. We will examine its timing and efficiency, the conformations of reassembled nucleosomes, and any preferential recycling to either daughter DNA.
This proposal places us in a unique position to make major contributions to the field of chromatin replication, and to provide the field with a powerful tool to investigate topics from fundamental questions in molecular biology to the performance of new cancer drugs.