Research

We use a variety of cellular, molecular, genomic, bio-informatic and modelling approaches to understand how DNA replication is regulated to ensure genome stability.

Our research areas

How do cells faithfully complete genome replication?


Our approach

We have developed and continue to develop cutting edge technologies.

The MinION from Oxford Nanopore Technologies can generate 10-20 Gb of DNA sequence in ultra-long reads (up to hundreds of kb long).

We use a variety of genomic methods to study DNA replication that take advantage of DNA high-throughput sequencing (HTS). First, hundreds of millions of short sequence reads allow us to ‘count’ DNA ‘tags’ at every location across the genome. For example, this allows us measure the change from one to two copies as each part of the genome replicates or which polymerase is responsible for replicating each DNA strand. Second, we have combined short and long read technologies to rapidly generate high quality de novo genome assemblies to facilitate our comparative genomics studies.

  1. Daigaku, Y, Keszthelyi, A, Müller, CA, Miyabe, I, Brooks, T, Retkute, R, Hubank, M, Nieduszynski, CA, Carr, AM. A global profile of replicative polymerase usage. Nat. Struct. Mol. Biol. 2015;22 (3):192-198. doi: 10.1038/nsmb.2962. PubMed PMID:25664722 PubMed Central PMC4789492.
  2. Müller, CA, Hawkins, M, Retkute, R, Malla, S, Wilson, R, Blythe, MJ, Nakato, R, Komata, M, Shirahige, K, de Moura, AP, Nieduszynski, CA. The dynamics of genome replication using deep sequencing. Nucleic Acids Res. 2014;42 (1):e3. doi: 10.1093/nar/gkt878. PubMed PMID:24089142 PubMed Central PMC3874191.
  3. Liti, G, Nguyen Ba, AN, Blythe, M, Müller, CA, Bergström, A, Cubillos, FA, Dafhnis-Calas, F, Khoshraftar, S, Malla, S, Mehta, N, Siow, CC, Warringer, J, Moses, AM, Louis, EJ, Nieduszynski, CA. High quality de novo sequencing and assembly of the Saccharomyces arboricolus genome. BMC Genomics. 2013;14 :69. doi: 10.1186/1471-2164-14-69. PubMed PMID:23368932 PubMed Central PMC3599269.
Stochastic mathematical models aid in the interpretation of whole genome replication timing data.

In collaboration with Alessandro de Moura we have developed stochastic mathematical models for chromosome replication. These models allowed us to discovered that stochastic replication origin activity leaves a ‘signature’ in population-based data. Application of our models to genomic data allow us to gain maximal biological insight, for example, revealing the efficiency of every origin and the distribution of replication termination events.

  1. Newman, TJ, Mamun, MA, Nieduszynski, CA, Blow, JJ. Replisome stall events have shaped the distribution of replication origins in the genomes of yeasts. Nucleic Acids Res. 2013;41 (21):9705-18. doi: 10.1093/nar/gkt728. PubMed PMID:23963700 PubMed Central PMC3834809.
  2. Retkute, R, Nieduszynski, CA, de Moura, A. Dynamics of DNA replication in yeast. Phys. Rev. Lett. 2011;107 (6):068103. doi: 10.1103/PhysRevLett.107.068103. PubMed PMID:21902372 PubMed Central PMC3671325.
  3. de Moura, AP, Retkute, R, Hawkins, M, Nieduszynski, CA. Mathematical modelling of whole chromosome replication. Nucleic Acids Res. 2010;38 (17):5623-33. doi: 10.1093/nar/gkq343. PubMed PMID:20457753 PubMed Central PMC2943597.
The synthetic yeast genome consortium (Sc2.0) aims to re-design and synthesize a 12 Mb designer yeast genome de novo.

In collaboration with Patrick Cai’s group, we are helping to re-design and characterise a synthetic yeast genome called Sc2.0. One of the design principles involves stripping out transposable element and moving tRNA genes. Both of these features are frequently co-located with early ‘firing’ replication origins. Therefore, we have been characterising the replication dynamics of synthetic chromosomes to test whether the co-localisation influences origin activation time. Furthermore, we have helped with the design of a tRNA neochromosome, for example, by providing non-native replication origins.

  1. Shen, Y, Wang, Y, Chen, T, Gao, F, Gong, J, Abramczyk, D, Walker, R, Zhao, H, Chen, S, Liu, W, Luo, Y, Müller, CA, Paul-Dubois-Taine, A, Alver, B, Stracquadanio, G, Mitchell, LA, Luo, Z, Fan, Y, Zhou, B, Wen, B, Tan, F, Wang, Y, Zi, J, Xie, Z, Li, B, Yang, K, Richardson, SM, Jiang, H, French, CE, Nieduszynski, CA, Koszul, R, Marston, AL, Yuan, Y, Wang, J, Bader, JS, Dai, J, Boeke, JD, Xu, X, Cai, Y, Yang, H. Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome. Science. 2017;355 (6329):. doi: 10.1126/science.aaf4791. PubMed PMID:28280153 PubMed Central PMC5390853.
One of our favourite organisms, the budding yeast Saccharomyces cerevisiae, gives us bread, wine, beer and the most powerful model organism.

We have made fundamental discoveries about how genomes replicate in bacteria, archaea and eukaryotes. In bacteria (Escherichia coli), we discovered that completion of genome replication has pathological potential. In archaea (Haloferax volcanii), we discovered the first example of a cellular genome able to replicate in the absence of replication origins, challenging the dogma that replication origins are necessary to coordinate chromosomal events. In yeasts (both fission and budding), we have discovered mechanisms regulating replication origin activity and that these mechanisms contribute to faithful chromosome inheritance. We are translating our technologies and findings from microbes to mammalian cells.

  1. Daigaku, Y, Keszthelyi, A, Müller, CA, Miyabe, I, Brooks, T, Retkute, R, Hubank, M, Nieduszynski, CA, Carr, AM. A global profile of replicative polymerase usage. Nat. Struct. Mol. Biol. 2015;22 (3):192-198. doi: 10.1038/nsmb.2962. PubMed PMID:25664722 PubMed Central PMC4789492.
  2. Hawkins, M, Malla, S, Blythe, MJ, Nieduszynski, CA, Allers, T. Accelerated growth in the absence of DNA replication origins. Nature. 2013;503 (7477):544-547. doi: 10.1038/nature12650. PubMed PMID:24185008 PubMed Central PMC3843117.
  3. Rudolph, CJ, Upton, AL, Stockum, A, Nieduszynski, CA, Lloyd, RG. Avoiding chromosome pathology when replication forks collide. Nature. 2013;500 (7464):608-11. doi: 10.1038/nature12312. PubMed PMID:23892781 PubMed Central PMC3819906.
  4. Siow, CC, Nieduszynska, SR, Müller, CA, Nieduszynski, CA. OriDB, the DNA replication origin database updated and extended. Nucleic Acids Res. 2012;40 (Database issue):D682-6. doi: 10.1093/nar/gkr1091. PubMed PMID:22121216 PubMed Central PMC3245157.
Replication origins are licensed during G1 phase by the loading of the MCM complex and activated during S phase.

How is DNA replication origin activity regulated?


DNA replication initiates at thousands of specific sites, called replication origins, found throughout the genome. Sufficient, appropriately distributed origins must be activated (‘fired’) to ensure faithful completion of genome replication. Our research aims to discover the mechanisms that the cell uses to regulate the firing of origins.

We have discovered that replication origin activity can be regulated by local DNA sequence elements (cis-regulation). For example, we found that proximity to the centromere results in early origin activation due to recruitment of the origin ‘firing’ factor, Dbf4. Disruption of this pathway results in delayed centromere replication and increased rates of chromosome loss.

  1. Hoggard, T, Shor, E, Müller, CA, Nieduszynski, CA, Fox, CA. A Link between ORC-origin binding mechanisms and origin activation time revealed in budding yeast. PLoS Genet. 2013;9 (9):e1003798. doi: 10.1371/journal.pgen.1003798. PubMed PMID:24068963 PubMed Central PMC3772097.
  2. Natsume, T, Müller, CA, Katou, Y, Retkute, R, Gierliński, M, Araki, H, Blow, JJ, Shirahige, K, Nieduszynski, CA, Tanaka, TU. Kinetochores coordinate pericentromeric cohesion and early DNA replication by Cdc7-Dbf4 kinase recruitment. Mol. Cell. 2013;50 (5):661-74. doi: 10.1016/j.molcel.2013.05.011. PubMed PMID:23746350 PubMed Central PMC3679449.
  3. Müller, CA, Nieduszynski, CA. Conservation of replication timing reveals global and local regulation of replication origin activity. Genome Res. 2012;22 (10):1953-62. doi: 10.1101/gr.139477.112. PubMed PMID:22767388 PubMed Central PMC3460190.
Activation of too few replication origins or double fork stalls can lead to incomplete chromosome replication.

What happens when replication is incomplete?


Failure to activate sufficient or appropriately distributed replication origins can result in unreplicated regions of the genome, which may not be faithfully segregated during cell division. For example, in mammalian cells chromosome common fragile sites have a paucity of active origins and a high frequency of chromosomal breaks and rearrangements due to failures during DNA replication. Therefore, we were surprised to discover that the halophilic archaea, Haloferax volcanii, can not only survive in the absence of DNA replication origins, but also has accelerated growth. Our ongoing research is addressing how eukaryotic cells control the distribution of active replication origins and the cellular response to failures to complete DNA replication. This research will help us understand normal development and ageing and how errors can lead to genetic variation and the onset of disease.

  1. Hawkins, M, Malla, S, Blythe, MJ, Nieduszynski, CA, Allers, T. Accelerated growth in the absence of DNA replication origins. Nature. 2013;503 (7477):544-547. doi: 10.1038/nature12650. PubMed PMID:24185008 PubMed Central PMC3843117.
  2. Newman, TJ, Mamun, MA, Nieduszynski, CA, Blow, JJ. Replisome stall events have shaped the distribution of replication origins in the genomes of yeasts. Nucleic Acids Res. 2013;41 (21):9705-18. doi: 10.1093/nar/gkt728. PubMed PMID:23963700 PubMed Central PMC3834809.
Recruitment of Dbf4-Cdc7 to the kinetochore promotes activation of proximal origins early in S phase.

What are the consequences of deregulated replication control?


It has long been known that chromosomes replicate in a characteristic and reproducible temporal order. This is dictated by the location and activity of the replication origins. The replication time of a DNA sequence correlates with gene expression, chromatin state, GC content, and subnuclear structure. However, it has remained unknown whether there is a physiological requirement for the regulation of replication timing. Our research is addressing whether it is important that a particular gene or DNA sequence replicates at a particular time during S phase.

We discovered that replication time is broadly conserved between closely related species, consistent with a physiological requirement for regulation of replication timing. More recently we have demonstrated that early replication of centromeres and certain genes is important for normal cell function. Failure to replicate centromeres early in S phase leads to increased levels of chromosome loss. Furthermore, replication in early S phase of certain genes, including histone genes, is required for their maximal expression in S phase. Our discoveries raise the intriguing possibility that the changes in replication time reported during carcinogenesis could affect gene expression and potentially contribute to disease progression.

  1. Müller, CA, Nieduszynski, CA. DNA replication timing influences gene expression level. J. Cell Biol. 2017;216 (7):1907-1914. doi: 10.1083/jcb.201701061. PubMed PMID:28539386 PubMed Central PMC5496624.
  2. Natsume, T, Müller, CA, Katou, Y, Retkute, R, Gierliński, M, Araki, H, Blow, JJ, Shirahige, K, Nieduszynski, CA, Tanaka, TU. Kinetochores coordinate pericentromeric cohesion and early DNA replication by Cdc7-Dbf4 kinase recruitment. Mol. Cell. 2013;50 (5):661-74. doi: 10.1016/j.molcel.2013.05.011. PubMed PMID:23746350 PubMed Central PMC3679449.
  3. Müller, CA, Nieduszynski, CA. Conservation of replication timing reveals global and local regulation of replication origin activity. Genome Res. 2012;22 (10):1953-62. doi: 10.1101/gr.139477.112. PubMed PMID:22767388 PubMed Central PMC3460190.

Our funding agencies

We are grateful for support from: