Publication highlights

This study defined the mechanism leading to critically short telomeres in the absence of RTEL1 and showed that telomerase, which extends telomeres in normal cells, is pathological when forks encounter an obstacle within the telomere. We showed that replication forks stall and reverse at persistent t-loops, which creates a pseudo-telomere substrate that is inappropriately stabilised by telomerase. Removing telomerase or blocking replication fork reversal rescued telomere dysfunction in Rtel1 deficient cells. We proposed that when persistent t-loops stall the replisome, telomerase inhibits fork restart, triggering the excision of the t-loop by SLX1/4 and loss of a substantial part of the telomere.

This study was the first to demonstrate that RAD51 paralogues bind to and structurally remodel the pre-synaptic RAD-51-ssDNA filament to a stabilised, “open”, and flexible conformation, which facilitates strand exchange with the template duplex. We showed that RAD51 paralogues act by binding the end of the presynaptic filament, which induces a conformational change that stabilises RAD-51 bound to ssDNA and primes the filament for strand exchange. These observations established for the first time the underlying mechanism of HR stimulation by Rad51 paralogues and revealed a new paradigm for the action of HR mediator proteins.

Evidence suggested that the telomere adopts a lasso-like t-loop configuration, which safeguards chromosome ends from being recognised as DNA double strand breaks. However, the regulation and physiological importance of t-loops in end-protection was uncertain. This study uncovered a phospho-switch in TRF2 that coordinates the timely assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response. These results were the first to definitively establish the t-loop as a physiologically important structure required to suppress checkpoint activation at telomere ends.

This work revealed that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues. In somatic cells, TRF2 sequesters the telomere within a t-loop, preventing telomere end-to-end fusions and inviability. In contrast, TRF2 is dispensable for telomere protection in pluripotent cells; ESCs lacking TRF2 grow normally, do not fuse their telomeres and form functional t-loops. Upon differentiation this unique attribute of stem cells is lost and TRF2 assumes its full role in end protection. The retention of end protection in the presence of t-loops, but absence of TRF2, confirmed that t-loops are a key mediator of telomere end protection irrespectively of how they form.

Like many developing tissues, the vertebrate neural tube is patterned by antiparallel morphogen gradients. Using quantitative gene expression and signalling measurements we derived and validated a characteristic decoding map that relates morphogen input to the positional identity of neural progenitors. This revealed a strategy that minimises patterning errors in response to the joint input of noisy opposing gradients. The study illustrates how we integrate quantitative data, developmental and microfluidic experiments with phenological and mechanistic models.

Despite evolutionarily conservation of molecular mechanisms, the speed of development varies substantially between species. Using in vitro directed differentiation of embryonic stem cells to motor neurons, we show that the programme of motor neuron differentiation runs twice as fast in mouse as in human. We provide evidence that a two-fold increase in protein stability and cell cycle duration in human cells compared to mouse can account for the slower pace of human development, indicating that global differences in kinetic parameters play a major role in interspecies differences in developmental tempo. This study establishes a new experimental system in which to address fundamental questions.

The prevailing view of neural induction in vertebrate embryos had been that cells are initially induced with anterior (forebrain) identity and then caudalising signals convert a proportion to posterior fates (spinal cord). Using chromatin accessibility, to define how cells adopt region-specific neural fates, combined with genetic and biochemical perturbations, we found that contrary to the established model, cells commit to a regional identity before acquiring neural identity. These findings prompt a revision to textbook models of neural induction. The study illustrates our adoption of new genomic methods (ATACseq) to address long-standing questions, and our capacity to productively collaborate with computational biologists.

Memory B cells (MBCs) are key for protection from reinfection. However, it is mechanistically unclear how germinal center (GC) B cells differentiate into MBCs. MYC is transiently induced in cells fated for GC expansion and plasma cell (PC) formation, so-called positively selected GC B cells. We found that these cells coexpressed MYC and MIZ1 (MYC-interacting zinc-finger protein 1 [ZBTB17]). MYC and MIZ1 are transcriptional activators; however, they form a transcriptional repressor complex that represses MIZ1 target genes. Mice lacking MYC-MIZ1 complexes displayed impaired cell cycle entry of positively selected GC B cells and reduced GC B cell expansion and PC formation. Notably, absence of MYC-MIZ1 complexes in positively selected GC B cells led to a gene expression profile alike that of MBCs and increased MBC differentiation. Thus, at the GC positive selection stage, MYC-MIZ1 complexes are required for effective GC expansion and PC formation and to restrict MBC differentiation. We propose that MYC and MIZ1 form a module that regulates GC B cell fate.

Memory B cells (MBCs) and plasma cells (PCs) are formed during the so-called germinal center (GC) B cell reaction. In the GC reaction B cells mutate their B cell receptor (BCR) genes and those that acquire a higher-affinity BCR for a pathogen antigen are presumably selected to survive and differentiate, whereas B cells carrying a lower-affinity BCR die. However, this cannot explain retention of GC B cells with varied BCR affinities and the formation of MBCs that normally carry lower-affinity BCRs. This work re-defines selection of GC B cells as permissive to ensure clonal diversity and broad protection.

D-cycloserine is an antibiotic used for decades to treat drug resistant tuberculosis. Its inhibition mechanism came into question when in a previous paper we determined alanine racemase activity in “fully inhibited” cells. This study demonstrated a previously unknown path during the assumed irreversible inhibition of alanine racemase that leads to the destruction of the antibiotic, meaning that alanine racemase is not irreversibly inhibited by the drug. The paper highlights the complexity of studying the chemical mechanisms of inhibition of enzymes and points to a novel strategy to design D-cycloserine analogues with improved properties.

Lentiviral IN proteins are notoriously poorly behaved in vitro, and the HIV 1 intasome has eluded structural biologists for over two decades. Prior research resulted in a collection of partial crystal and NMR structures that did not explain how lentiviral integrase synapses viral DNA ends. This paper described the first structure of the lentiviral intasome, solving the long-standing mystery and reconciling years of HIV-1 integrase structural biology and biochemistry.

Here, we described a cryo-EM structure of a retroviral intasome in a functional complex with a nucleosome. The structure revealed a multivalent interface of the viral integration machinery and chromatin, involving both gyres of nucleosomal DNA and histones. Whilst the histone octamer remains intact, the DNA is lifted from its surface to allow for strand transfer at highly preferred integration sites. These data provided a unique snapshot of an enzyme recognizing and acting upon nucleosomal DNA. The structure was the first to illustrate nucleosome flexibility facilitating a biological process and, as such, had far-reaching implications for chromosome biology.

HIV integrase inhibitors represent some of the most impactful antimicrobial inhibitors. The second-generation drugs display improved barriers to the emergence of resistance, which spearheaded their worldwide rollout. Yet not even the most advanced compounds are immune to viral resistance. Our results explained the mechanism of viral resistance associated with the most common drug resistance mutations. Furthermore, we established the key difference between the first and second-generation strand transfer inhibitors, which will inform further development of this drug class.

A team led by the Cherepanov lab has found a molecule that can block the binding of a subset of human antibodies to SARS-CoV-2. This could explain patients who, despite having high levels of antibodies, become ill.

Oesophageal adenocarcinoma shows high genetic heterogeneity making the identification of cancer drivers challenging. We developed a machine learning algorithm to identify cancer drivers in 261 oesophageal adenocarcinomas. Although most predicted drivers were rare or patient-specific, they all perturbed well-known cancer pathways. Using the recurrence of the same pathway perturbations rather than individual genes, we stratified patients into six groups different for their clinical features. We validated experimentally the contribution of these genes to disease progression and revealed acquired dependencies exploitable in therapy. This study described a new way to identify cancer drivers that we have recently further developed for application in precision oncology.

The MCM replicative helicase is loaded onto duplex DNA as a double hexamer. Here we use time-resolved cryo-EM to show that ORC binds to its high affinity binding site to load the first MCM hexamer. ORC then releases this site and it, or another ORC molecule then binds the B2 element, which contains a degenerate ORC binding site. This binding is stabilised by a novel interaction between the Orc6 subunit of ORC and the N-terminus of the MCM hexamer. ORC then recruits and loads the second hexamer by the same mechanism as the first hexamer. We employed newly developed in silico reconstitution approaches to describe the full context of the helicase loading reaction, studied on a near-native, chromatinised origin of replication. This study radically changes our approach to investigating chromosome replication with cryo-EM.

This paper decribes how we were able to successfully repurpose the Crick to increase the capacity for Sars-CoV-2 testing in unpredented times.

This important paper showed very high levels of infection amongst healthcare workers in a local hospital. It has influenced government policy – asymptomatic healthcare workers are to be screened as per our recommendation (announced October 12th).