Single molecule detection of DNA replication errors

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. Our research aims to determine how cells ensure that the replication of each chromosome is completed accurately. 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. We will apply this approach to determine what DNA sequences can pose an obstacle to the DNA replication machinery; what protein factors assist in getting past such obstacles; and how pausing of the replication machinery is linked to errors during the copying process. This is important because a single DNA replication error on one chromosome in a single cell division can give rise to genomic disorders, including cancer.

Complete, accurate genome replication is essential for life. Our long-term goal is to determine how cells faithfully complete genome replication. Errors in DNA replication occur on single molecules in individual cells; however these errors are hidden from view in genomic approaches that look at data from populations of several million cells. Recently, we developed the first single molecule DNA sequencing method 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 initiation, termination and fork pausing sites on single-molecules genome-wide. The sensitivity of our single molecule approach will allow us to quantitatively identify and characterise (in vivo) obstacles to DNA replication and how they contribute to genome instability.

The pausing of a DNA replication fork leads to the accumulation of fragile, single stranded DNA that is prone to base damage, fork slippage and double strand breaks. Therefore, fork pausing is a major source of replicative errors, including point mutations, expansion/contraction of repeats, deletions and translocations. First, our single molecule approach will allow us to quantitatively determine the location and duration of replication fork pausing sites throughout the genome. This will systematically determine the nature of naturally occurring 'difficult-to-replicate' sequences. Second, we will quantify the role of accessory proteins that support replication through difficult-to-replicate sequences. Third, we will determine the barrier that short tandem repeats pose to stable DNA replication, both at the level of replication fork progress and repeat copy number stability. Together these experiments will provide the first high-resolution, whole-genome view of DNA replication fork progression on single molecules.