Unpicking human DNA replication with single molecules
About Rosemary Wilson
Rosemary completed her Ph.D. with Dawn Coverley at the University of York working on the temporal and spatial organisation of DNA replication, and the role of initiating factor CIZ1. She then moved to Cath Green’s lab at the University of Oxford to investigate the molecular mechanisms underlying PCNA-Associated Repair Disorder, caused by a rare mutation in the DNA replication factor PCNA. Rosemary is currently working with Conrad Nieduszynski where she uses single molecule sequencing and other genomic methods to study DNA replication dynamics.
Single molecule methods are a powerful tool for the study of biological processes with significant heterogeneity. One such process is DNA replication in human cells, and the poorly understood changes in replication timing and origin usage that occur during development and disease. Previously, in yeast, we described the measurement of replication dynamics by detecting nucleotide analogues in long single molecules (D-NAscent). We now present replication dynamics on single molecules across the human genome. Synchronised human cells were pulsed with BrdU to label nascent DNA, which is then detected by nanopore sequencing (Oxford Nanopore MinION) and D-NAscent software. The patterns of DNA replication that we detect, both genome wide and using targeted enrichment, reveal common and rare replication events that inform on the process and regulation of genome replication. This provides a powerful new tool for the analysis of human genome replication dynamics and mechanisms that underlie diseases.