Image: National Centre for X-ray Tomography

Published highlights

Replication fork arrest in a palindrome

• Mizuno et al; (2009) Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism. Genes Dev. 23, 2876-2886.
• Mizuno et al (2012) Recombination-restarted replication makes inverted chromosome fusions at inverted repeats. Nature, 493, 246-249

Gene amplification plays important roles in the progression of cancer and also contributes to acquired drug resistance during cancer treatments. Amplification is proposed to frequently initiate via dicentric palindromic chromosome production and subsequent breakage-fusion-bridge cycles. Using a variant of our replication fork arrest system in fission yeast, we were able to demonstrate that acentric and dicentric palindromic chromosomes formed via a homologous recombination protein-dependent fusion of nearby inverted repeats in response to replication forks arrest within the inverted repeats. Our genetic and molecular analyses demonstrated that these acentric and dicentric chromosomes arose not by any previously described mechanism, but through a replication template exchange mechanism that does not involve the expected DNA double-strand break. We were thus able to propose an alternative mechanism for the generation of palindromic chromosomes that is dependent on replication forks arresting within closely spaced inverted repeats.

During our work with the palindrome system we noticed that gross chromosomal rearrangment events were greatly elevated when compared to a non-palindromic system. This lead us to try and understand why this was. We found that the consequence of replicating a palindromic DNA sequence with a fork that had been restarted by homologous recombination caused a novel “U-turn” at the centre of the palindrome or closely-spaced inverted repeat. This defined a new mechanism of chromosomal rearrangement to which recombination-restarted forks are highly susceptible to (up to 1 in 40 replication events). We thus propose that the error-prone nature of restarted forks contributes to the generation of GCRs and gene amplification in cancer, and to non-recurrent Copy Number Variations in genomic disorders.

Characterisation of the replication fork

• Miyabe et al (2015) Polymerase δ replicates both strands after homologous recombination-dependent fork restart. Nat Struct Mol Biol. 22, 932-938.

Because fork inactivation is common, passive convergence of an adjacent fork is insufficient to rescue all inactive forks. Thus, eukaryotic cells have evolved homologous recombination-dependent mechanisms to restart persistent inactive forks. Completing DNA synthesis via homologous recombination-restarted replication  ensures cell survival, but at a cost. One such cost is increased mutagenesis because HR-initiated replication is more error prone than canonical replication. This increased error rate implies the mechanism is distinct from that of a canonical fork. In S. cerevisiae when replication is restarted from a DNA double strand break  (DSB) in mitotically arrested cells "Break induced replication" proceeds via a migrating D-loop and is conservative (i.e. both newly synthesised strands are in the same duplex). Using our RTS1 system we showed that, when restating a fork by HR in S phase (and without a DSB) the resulting replication fork is semi conservative, but both the leading and lagging strands are synthesized by DNA polymerase δ. 

A non-canonical view of PCNA ubiquitylation

• Daigaku wt al (2017) PCNA ubiquitylation ensures timely completion of unperturbed DNA replication in fission yeast. PLoS Genet. 13:e1006789.

PCNA ubiquitylation on lysine 164 is required for DNA damage tolerance. In many organisms PCNA is also ubiquitylated in unchallenged S phase but the significance of this had not been established. Using Schizosaccharomyces pombe, we demonstrated that lysine 164 ubiquitylation of PCNA contributes to efficient DNA replication in the absence of DNA damage. Loss of PCNA ubiquitylation manifests most strongly at late replicating regions and increases the frequency of replication gaps. We showed that PCNA ubiquitylation increases the proportion of chromatin associated PCNA and the co-immunoprecipitation of Polymerase δ with PCNA during unperturbed replication. We propose that ubiquitylation acts to prolong the chromatin association of these replication proteins to allow the efficient completion of Okazaki fragment synthesis by mediating gap filling.

Methods development

• Watson et al (2008) Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe. Gene, 407: 63-74.
• Watson et al (2011) Regulation of gene expression at the fission yeast Schizosaccharomyces pombe urg1 locus. Gene. 483, 75-85.

We have continued our tradition on developing new research tools for S. pombe and presenting these to the community via publication:

We developed a method to rapidly generate chromosomal mutations in S. pombe genes. This has two specific advantages: it allows rapid and efficient mutagenesis studies and it allows iterative gene tagging. This method, Recombinase Mediated Cassette Exchange (RMCE) is based on a cassette consisting of the S. pombe ura4⁺ selectable marker that is flanked by a wild-type loxP site at one end and by a modified heterospecific lox site (loxM3) at the other end. The cassette is stable because these two lox sites cannot recombine with each other. Initially, the cassette is integrated at the chosen chromosomal locus by classical HR. Subsequently, rapid exchange is achieved by introducing a Cre-expression plasmid containing an equivalent cassette harbouring the required tag or modified gene sequence. We established a range of reagents and verified the systems utility. Subsequently, we have been able to use this system to contribute to several collaborative studies (i.e. Lloyd et al 2009, A super-modular FHA/BRCT-repeat architecture mediates Nbs1 adaptor function in response to DNA-damage. Cell, 139, 100-111).

We have developed a rapid and efficient inducible promoter system for fission yeast that utilises the expression of proteins from the native urg1 locus. The lack of a rapid induction system for S. pombe that is equivalent to the GAL system in S. cerevisiae has been a serious limitation in the field. We adapted our Cre-lox RMCE system to allow efficient replacement of the urg1 ORF by any gene of interest. Previous work (Watt et al, 2008, PLoS One. 2008. 16, e1428) identified urg1 as rapidly induced by uracil addition to the growth medium, but it was subsequently found that, when the promoter was introduced into another sequence context, induction was largely abolished because the “off level” increased ~ 8-10 fold, preventing a functional “off” phenotype for all of the genes tested. We have characterised induction from our modified urg1 locus as ~x80 with 30-60 minutes and have used the system to establish an HO-single strand annealing system where efficient cleavage can be achieved within 60 minutes. A single plasmid cloning event, followed by a plasmid transformation that results in cassette exchange into the modified urg1 locus now allows any gene to be regulated in this way.

• Etheridge et al (2014) Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy. Nucleic Acids Res. 42:e146.

We have developed a photo-activated localization microscopy-based method to directly visualize and quantify DNA-associated proteins in unfixed eukaryotic cells. The methodology exploits long exposure times (motion blurring) to distinguish between diffusing molecules and those that are restricted by DNA association. We validate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins using a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA.