• Research

Exploring the impact of DNA replication on genome evolution and stability.

Single molecule analysis of genome replication

Project summary.

All cells contain a complete copy of the organism's DNA, the genetic blueprint of life, packaged into discrete units called chromosomes. Since new cells need a copy of the genetic material, the chromosomes must be completely and accurately replicated before the cell can divide. Mistakes during DNA replication fuel evolution, but can also drive disease. This research aims to determine how cells ensure that the replication of each chromosome is completed accurately.

Problems during DNA replication may underlie or be diagnostic for some diseases. Problems can occur when the machinery that copies the DNA encounters an obstacle. This can cause the DNA replication machinery to slow or pause which in turn can give rise to duplications, the expansion/contraction of repeated sequences or even lead to breaks in both strands of the DNA. Therefore, although obstacles rarely cause a problem for DNA replication, when they do the consequences can be catastrophic for the cell.

Rare events, such as pausing of the replication machinery, can be difficult to detect, since most DNA replication is occurring normally. These rare, but serious events, present a ‘needle in a haystack’ problem for researchers. We have developed a high-throughput DNA sequencing technology that allows us to study the kinetics of DNA replication in vivo on single molecules. This technology allows us to rapidly search for the ‘needle in the haystack’ and identify rare, but serious, events such as the slowing down or pausing of the DNA replication machinery. This is important because a single DNA replication error on one chromosome in a single cell division can give rise to disease.

Details.

Recently, we developed the first single molecule DNA sequencing technology for the study of genome replication (D-NAscent) that can detect important events hidden in population data. D-NAscent uses nanopore sequencing to detect base analogues incorporated into DNA on extremely long reads.

The pattern of incorporated analogue reveals replication initiation, termination and fork pausing sites on single-molecules genome-wide. We are applying this technology in yeast and human cells to generate the first high-resolution, whole-genome view of DNA replication fork progression on single molecules. These data will allow us to identify aberrant patterns of DNA replication that may drive genome evolution and the development of disease.

Tools.

DNA Replication Origin (OriDB) databases

Databases of DNA replication origin sites in budding and fission yeast.

Genomic data

Our published genome-wide data can be downloaded from NCBI GEO.

Software and computational pipelines

Our published software and computational pipelines are shared via GitHub.

Publications.

Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads

Müller, C., Boemo, M., Spingardi, P., Kessler, B., Kriaucionis, S., Simpson, J., Nieduszynski, C. (2019).  Nature Methods 16(5), 429-436.

Impact statement.

Faithful DNA replication is fundamental to the survival of every organism. Mistakes during DNA replication are the major source of genetic variation that underlies disease and the acquisition of novel traits (beneficial and detrimental). Changes in the copy number of short tandem repeat sequences can occur when replication forks pause, with dramatic consequences.

For example, repeat variability within genes and promoters are associated with important traits, including salmon migration, the circadian clock in Drosophila, microbial drug resistance and human disease associated gene expression. Furthermore, triplet expansion is associated with a number of human diseases, including myotonic dystrophy and Huntington's disease.

Therefore, the identification of aberrant patterns of DNA replication is crucial to understand the genomic changes that fuel evolution and drive disease. 

Related reading.