Biology subjects

Carr, A. M.

Publications and source records attributed to Carr, A. M..

6 recordsLinked to original sources

bioRxiv2025

Single-molecule sequencing maps replication dynamics across the fission yeast genome, including centromeres

DNA replication dynamics in fission yeast remain incompletely understood, particularly in repetitive regions such as the centromeres and the rDNA. Here, we establish DNAscent--a nanopore sequencing method that detects BrdU-labelled nascent DNA and infers replication dynamics--to map replication at single-molecule resolution in fission yeast. For the first time, we have identified thousands of replication forks, as well as initiation, termination and pause events, on single sequenced molecules across the whole genome. This high coverage allowed us to identify replication patterns in poorly characterised regions. In the rDNA, we detect strand-specific pausing at replication fork barriers. At the mating-type locus, we find the most frequent pause site outside the rDNA. At centromeres, we find that replication initiation predominantly occurs in the outer repeats, while termination localises to central regions and that, only in centromere 2, there is an enrichment in pauses at the centromeric tRNAs. This work establishes a powerful single-molecule method for studying replication dynamics in fission yeast and provides insights into replication across repetitive regions that constitute a significant portion of the genomes of more complex organisms.

genomics↗

bioRxiv2021

Global landscape of replicative DNA polymerase usage in the human genome

The division of labour among DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequence (Pu-seq) in HCT116 cells and mapped Pol{varepsilon} and Pol usage genome wide. The polymerase usage profiles show Pol{varepsilon} synthesises the leading strand and Pol contributes mainly to lagging strand synthesis. Combining the Pol{varepsilon} and Pol profiles, we accurately predict the genome-wide pattern of fork directionality plus zones of replication initiation and termination. We confirm that transcriptional activity contributes to the pattern of initiation and termination and, by separately analysing the effect of transcription on both co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands in both co- directional and convergent forks is compromised by transcription. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability.

molecular biology↗

bioRxiv2020

Single-molecule live cell imaging of the Smc5/6 DNA repair complex

The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the ssDNA binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.

molecular biology↗

bioRxiv2019

Biological network topology features predict gene dependencies in cancer cell lines

In this paper we explore computational approaches that enable us to identify genes that have become essential in individual cancer cell lines. Using recently published experimental cancer cell line gene essentiality data, human protein-protein interaction (PPI) network data and individual cell-line genomic alteration data we have built a range of machine learning classification models to predict cell line specific acquired essential genes. Genetic alterations found in each individual cell line were modelled by removing protein nodes to reflect loss of function mutations and changing the weights of edges in each PPI to reflect gain of function mutations and gene expression changes.\n\nWe found that PPI networks can be used to successfully classify human cell line specific acquired essential genes within individual cell lines and between cell lines, even across tissue types with AUC ROC scores of between 0.75 and 0.85. Our novel perturbed PPI network models further improved prediction power compared to the base PPI model and are shown to be more sensitive to genes on which the cell becomes dependent as a result of other changes. These improvements offer opportunities for personalised therapy with each individuals cancer cell dependencies presenting a potential tailored drug target.\n\nThe overriding motivation for predicting cancer cell line specific acquired essential genes is to provide a low-cost approach to identifying personalised cancer drug targets without the cost of exhaustive loss of function screening.

systems biology↗

bioRxiv2019

S. pombe DNA translocases Rrp1 and Rrp2 have distinct roles at centromeres and telomeres that ensure genome stability.

Homologous recombination (HR) is a DNA repair mechanism that ensures, together with heterochromatin machinery, the proper replication, structure and function of telomeres and centromeres that is essential for the maintenance of genome integrity. Schizosaccharomyces pombe Rrp1 and Rrp2 participate in HR and are orthologues of Saccharomyces cerevisiae Uls1, a SWI2/SNF2 DNA translocase and SUMO-Targeted Ubiquitin Ligase. We show that Rrp1 or Rrp2 upregulation leads to chromosome instability and growth defects. These phenotypes depend on putative DNA translocase activities of Rrp1 and Rrp2. Either Rrp1 or Rrp2 overproduction results in a reduction in global histone levels, suggesting that Rrp1 and Rrp2 may modulate nucleosome dynamics. In addition we show that Rrp2, but not Rrp1, acts at telomeres. We propose that this role depends on the previously described interaction between Rrp2 and Top2. We conclude that Rrp1 and Rrp2 have important roles for centromere and telomere function and maintenance, contributing to the preservation of genome stability during vegetative cell growth.\n\nSUMMARY STATEMENTSchizosaccharomyces pombe DNA translocases Rrp1 and Rrp2 modulate centromere and telomere maintenance pathways and dysregulation of their activity leads to genome instability.

cell biology↗

bioRxiv2018

An essential role for dNTP homeostasis following CDK-induced replication stress

Replication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here we show that CDK-induced replication stress is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, Mus81-dependent DNA damage, and to genome instability. Cells respond to this replication stress by increasing dNTP supply through Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 tri-methylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a dNTP supply and demand model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.

molecular biology↗