Publication highlights

Long DNA molecules form the basis of our lives, and these must be intricately packed into chromosomes to be passed on from one cell generation to the next.

Key to DNA compaction is a ring-shaped protein complex called condensin. How condensin achieves this miraculous task remained mysterious. A popular idea is that condensin extrudes DNA loops, much like threading pieces of string through the eye of a needle. However, chromosomal DNA exists in a form that is likely too bulky to slide through the eye. In search for an alternative mechanism, Crick researchers made pure condensin and examined how it engages with DNA in a test tube. This approach revealed the striking ability of condensin to sequentially entrap two DNA molecules that find each other by pure chance. The researchers even watched this process happen in front of their own eyes using a specialized microscope. Collaborators from Hokkaido University in turn found compelling evidence for the new capture mechanism inside chromosomes of living cells. Together, these results change our way of thinking about chromosomes: condensin stitches up pieces of DNA to weave the fabric of a chromosome.

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.

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).

Here we showed that Sld3, which we previously identified as being one of two essential cyclin dependent kinase (CDK) substrates in replication, is a phosphopeptide binding protein which binds specifically to Mcm4 and Mcm6 when they have been phosphorylated by Dbf4 dependent kinase (DDK). Sld3 then directly recruits Cdc45 to MCM and, via CDK phosphorylation, recruits the remaining firing factors. We had previously shown that Sld3 is also one of two targets of the DNA damage checkpoint kinase involved in inhibiting origin firing in response to DNA damage. Thus, Sld3 plays key roles with all three kinases that regulate replication (CDK, DDK, Rad53).

This paper follows on from our work on WAC and the role of centrosomes in autophagy. We discovered an important centriolar protein has a specific motif (LIR motif) enabling its binding to a key autophagy protein. In collaborative work, we determined the structure and the important features of the LIR motif, and extended the findings to a group of autophagy proteins to provide an important advance on our understanding of selective autophagy. I chose this work because it is a tour de force of structure and biochemistry and a very substantial collaboration between Structural Biology and Peptide Chemistry STPs

Unlike kinases, PPP-family phosphatases such as PP1 have little intrinsic specificity. PP1 acts in partnership with over 200 different PP1-interacting proteins, but it has remained unclear how they might confer sequence-specificity on PP1. We used proteomics to identify dozens of candidate Phactr1/PP1 substrates, and used structural and biochemical approaches to show that the Phactr1/PP1 holoenzyme is sequence-specific. Phactr1 binding reshapes the PP1 hydrophobic groove, thereby creating a novel composite hydrophobic surface for substrate recognition. This study explains how cofactors can enhance the reactivity of PP1 toward specific substrates, and suggests a way forward for the development of PP1 holoenzyme-specific inhibitors.

This paper shows that the ArhGAP9/12/15/27 and ArhGAP32/33 families of rhoGAPs are RPEL proteins whose activity is coupled to G-actin concentration. G-actin forms a 1:1 complex with these ArhGAPs, interacting with an RPEL motif located between the PH and GAP domains, thereby inhibiting their GAP activity. Mutations that block G-actin binding exhibit elevated GAP activity towards their substrate GTPases Rac and Cdc42. Strikingly, treatment of cells with drugs enhancing or inhibiting G-actin/ArhGAP interaction has corresponding effects on Rac GTP loading. These results establish a novel homeostatic feedback loop, in which ArhGAP12-family (and presumably ArhGAP32-family) GAP activity increases when G-actin levels become limiting.

Building on our successful biochemical reconstitution of topological cohesin loading onto DNA, we completed the reconstitution of both dynamic loading as well as unloading. We realised that both loading and unloading follow a very similar trajectory through sequential ATPase and kleisin gates, only one of which can be open at any one time. This formed the basis for our unified DNA passage proposal both into and out of the ring.

Cohesin is a ring-shaped protein complex that topologically entraps DNA to fulfil key functions in chromosome architecture. In a collaborative and multidisciplinary approach, we used cryo-EM, biochemical and biophysical techniques to describe how ATP-fuelled structural changes of the cohesin complex drive the DNA entry reaction into the cohesin ring. This solves one of the outstanding riddles in molecular biology.