Publications

Discovering where DNA replication initiates in human cells is crucial for understanding how cells copy their genetic information. Our study explores where and how often DNA replication begins across the human genome. To achieve this, we employed a technique using in vivo BrdU incorporation followed by detection by single-molecule nanopore sequencing. We tested various BrdU concentrations on cultured human cells, sequenced the resulting DNA, and developed a computational method to identify replication initiation events from the sequencing data. We discovered that a large proportion of DNA replication begins outside known initiation zones, demonstrating a dispersed initiation pattern across the genome. We concluded that while a subset of initiation events occurs in focused zones and is influenced by factors such as transcription, the majority are dispersed and not associated with specific sequence features or epigenetic marks. This understanding refines the current model of how DNA replication is orchestrated within human cells and could inform studies on genome stability and the onset of diseases like cancer.

Discovering where DNA replication initiates in human cells is crucial for understanding how cells copy their genetic information. Our study explores where and how often DNA replication begins across the human genome. To achieve this, we employed a technique using in vivo BrdU incorporation followed by detection by single-molecule nanopore sequencing. We tested various BrdU concentrations on cultured human cells, sequenced the resulting DNA, and developed a computational method to identify replication initiation events from the sequencing data. We discovered that a large proportion of DNA replication begins outside known initiation zones, demonstrating a dispersed initiation pattern across the genome. We concluded that while a subset of initiation events occurs in focused zones and is influenced by factors such as transcription, the majority are dispersed and not associated with specific sequence features or epigenetic marks. This understanding refines the current model of how DNA replication is orchestrated within human cells and could inform studies on genome stability and the onset of diseases like cancer.

This work is H1 recommended.

During S phase, the relative DNA copy number increases for each region as it is replicated. Therefore, measuring DNA copy number can be used as a proxy for replication time. Here we present a step-by-step protocol to determine genome replication dynamics using high-throughput DNA sequencing to measure DNA copy-number. Cell populations can be obtained in three variants of the method sort-seq, sync-seq and MFA-seq.
We have shared our computational pipeline and example sequence data - see the resources page.

This work is H1 recommended.

DNA replication initiation is stochastic and obscured in population datasets. In this paper we introduce D-NAscent, a method to detect base analogs in ultra-long nanopore sequencing reads. This allowed us to identify replication origins and replication fork movement genome-wide on sequence reads of 20–160 kilobases. Our study marks the first, whole genome characterisation of DNA replication dynamics on single-molecules.
We have shared both the D-NAscent software and underlying sequence data - see the resources page.
This work is H1 recommended.

In this study we address the long-standing question - do topologically associating domains (TADs) act as a causative organizing structure for replication timing domains? Using rapid and complete cohesin degradation we observe no perturbations to replication timing. Our results suggest that cohesin-mediated genome architecture is not required for replication timing patterns.

In this paper we describe the use of digital PCR to precisely and rapidly measure DNA copy number as a proxy for replication time. As a proof-of-principle we demonstrate the method’s utility for the study of DNA replication time in both yeast and human cells. We show the power of the method by undertaking a targeted screen of kinetochore mutants to discover those that affect centromere replication time. As part of this work we generated whole genome DNA replication timing data (sort-seq) for cultured human cells (HeLa).
These data can be visualised via the link to our UCSC genome browser hub on the resources page.

Chromosomes replicate in a characteristic and reproducible temporal order. However, it has been unknown whether there is a physiological requirement for the regulation of replication timing. In this paper, we report our discovery that early DNA replication is required for the highest expression levels of certain genes.
Highlighted in J. Cell Biol. by María Gómez, selected for the Nuclear biology special collection by Ana Pombo and selected for the DNA replication and genome instability special collection by Agata Smogorzewska.
Selected as “of outstanding interest” by Mirit Aladjem and colleagues in their Current Opinion in Cell Biology review.

This work is H1 recommended.

This collaborative study reveals that polymerase epsilon is responsible for leading strand synthesis, with polymerase delta undertaking lagging strand synthesis. Our polymerase usage data directly maps replication origin location, quantifies origin efficiency, reveals that replication termination events are dispersed across large termination zones and indirectly estimates replication timing.
With associated News & Views by Sue Jinks-Robertson & Hannah L Klein.

In this study we used a combination of high-resolution, genomic-wide DNA replication data, mathematical modelling and single cell experiments to reveal the stochastic of replication initiation and termination. We find that the homogeneous picture implied by ensemble datasets masks biologically significant cell-to-cell variation in genome replication.

We discovered that the halophilic archaea, Haloferax volcanii, not only can survive in the absence of DNA replication origins, but also has accelerated growth.
This work is H1 recommended and featured in a research highlight in Nature Reviews Microbiology.
See associated article at The Conversation.

This paper describes and rigorously validates a range of next generation sequencing approaches to reveal at unprecedented resolution the temporal order of genome replication. My research group and others are applying these sequencing approaches to a range of questions in a variety of organisms.

We discovered that kinetochore recruitment of the Dbf4-dependent kinase ensures early centromere replication. Mutations that delay centromere replication give rise to elevated chromosome loss, revealing the first physiological role for temporal regulation of genome replication.
This work is H1 recommended.

We found that the temporal order of genome replication is conserved between species. The discovery of a minority of DNA replication origins that have evolved different activities allowed us to demonstrate that origin activity is locally (cis) regulated.

This work is H1 recommended.

This work is H1 recommended.

We present a mathematical model for whole chromosome replication, conceptualising how key molecular properties of DNA replication are related and demonstrating the wealth of information available in quantitative genomic datasets.
This work is H1 recommended.

This work is H1 recommended.

This paper presents the DNA replication origin database, OriDB, a catalogue of confirmed and predicted DNA replication origin sites. This database is an example of our commitment not only to sharing data, but also to making it highly accessible to the research community – see links on the resources page.

This was the first paper to use comparative genomics to investigate DNA replication. We discovered that the functional sequence elements at DNA replication origins are evolutionarily conserved, leaving a phylogenetic footprint that allowed them to be pin- pointed genome-wide.
This work is H1 recommended.