Newly copied sister chromatids are tethered together by the cohesin complex, but how sister chromatid cohesion coordinates with DNA replication is poorly understood. Prevailing models suggest that cohesin complexes, bound to DNA before replication, remain behind the advancing replication fork to keep sister chromatids together. By visualizing single replication forks colliding with preloaded cohesin complexes, we find that the replisome instead pushes cohesin to where a converging replisome is met. Whereas the converging replisomes are removed during DNA replication termination, cohesin remains on nascent DNA and provides cohesion. Additionally, we show that CMG (CDC45–MCM2-7–GINS) helicase disassembly during replication termination is vital for proper cohesion in budding yeast. Together, our results support a model wherein sister chromatid cohesion is established during DNA replication termination.

Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.

The cohesin complex tethers sister chromatids together from the moment they are generated in S-phase until their separation in anaphase 1,2 . This fundamental phenomenon, called sister chromatid cohesion, underpins orderly chromosome segregation. The replisome complex coordinates cohesion establishment with replication of parental DNA 3 . Cohesion can be established by cohesin complexes bound to DNA before replication 4,5 , but how replisome interaction with pre-loaded cohesin complexes results in cohesion is not known. Prevailing models suggest cohesion is established by replisome passage through the cohesin ring or by transfer of cohesin behind the replication fork by replisome components 5 . Unexpectedly, by visualising single replication forks colliding with pre-loaded cohesin complexes, we find that cohesin is pushed by the replisome to where a converging replisome is met. Whilst the converging replisomes are removed during DNA replication termination, cohesin remains on nascent DNA. We demonstrate that these cohesin molecules tether the newly replicated sister DNAs together. Our results support a new model where sister chromatid cohesion is established during DNA replication termination, providing important insight into the molecular mechanism of cohesion establishment.

A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits 1 . Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 Å resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the α-helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching reveals that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 results in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.

The Biochemical Society held its Strategy Retreat at the end of November 2020 and, at this meeting, our Council of Trustees considered the key opportunities and challenges in the year ahead (find more on the outcomes here). Following our earlier commitments to represent all members of the community regardless of career stage, it was noted that EDI (equality, diversity and inclusivity) needs to be considered as part of everything we do and we are committed to improving ‘in-house’ diversity across our committees, in parallel to developing our EDI strategy further. Part of this commitment includes representation of those at all career stages, from students through early career researchers (ECRs) to established scientists.Portland Press is the wholly owned publisher for the Biochemical Society, publishing seven journals as well as The Biochemist, the freely available magazine which acts as the voice of the Society to the community. Roughly a third of the Society’s members are early career molecular bioscientists. We are delighted to be able to share with you some of our recent activities aimed at supporting and working with ECRs.The Early Career Advisory Panel (ECAP) was established in November 2018 and is currently composed of 10 ECR representatives from industry and academia, and myself (Dominika) as the Chair. ECAP discusses issues related to ECRs, represents the views of ECRs to the Biochemical Society (with the Chair of the Panel also being a Trustee of the Society) and offers advice so that the concerns and needs of this section of the community can feed into the Society’s strategic objectives. The ECAP aims to shape the way the Society achieves its mission by engaging with ECRs in all areas of the Society’s mandate, such as communication, policy and training.The strategy of the Biochemical Society is set and driven by its Council of Trustees, which is elected by members and has the ultimate responsibility for all aspects of the Society’s activities. Responsibility for specific areas of activity and function is delegated to its sub-committees, with publishing activities being the responsibility of Publications Committee and Portland Press Board. During 2020, both Publications Committee and Portland Press Board thought that it would be valuable to hear the views of ECRs on policy and publishing items that were being discussed within the publishing landscape – by the end of the year, the ECR Taskforce was launched to enable us to do just that.The aim of the taskforce is to discuss and advise on potential publishing activities and policies, specifically in the context of how these might impact ECRs, and to give input into how the scholarly publishing ecosystem could/should develop (over the next 10 years) to support ECRs in their career progression.The taskforce of approximately 30 ECRs working in the biosciences met virtually in early February 2021 to discuss the topic of peer review and preprints. Discussions included the impact of a journal’s peer review model when choosing to submit to or review for that journal; the importance of recognition for peer review work; and whether preprints are a ‘friend or foe’ of journal publishing. There was excellent discussion among the groups, with forms of reviewer recognition being particularly important to the group. Key points have been fed back to the Society’s Publications Committee and Portland Press Board to feed into their own discussions on peer review and preprints.In May 2021, the taskforce met again, this time to discuss authorship criteria and publication metrics. The taskforce was asked which metrics they considered most important for their own personal development as well as those used to assess a journal, with several factors from time to decision, previous experience, costs and citations all being mentioned. Portland Press will use this feedback to consider the information currently presented to users to ensure that those of most value are being surfaced. The taskforce were also asked their views on authorship, and while there was consensus that to be a named author one must have ‘contributed significantly’ to a paper, there was discussion over the various forms that might take and how to standardize them. The taskforce noted that the CRediT system, recently introduced for submissions to the journals published by Portland Press, was a positive step in making author contributions more quantifiable.Recognizing that editorial work is something many ECRs are interested in getting more involved in, in 2021 Portland Press launched a pilot Editorial Board Mentorship Scheme aimed at ECRs. The aim of the scheme is to pair up ECRs with existing Associate Editors on our journals and to facilitate a 1:1 mentorship programme which will introduce the ECR to the role and responsibilities involved in academic publishing.This scheme is not intended to provide training in acting as a reviewer on a paper, but to go deeper – we are aiming to give the ECRs the skills needed to join an editorial board in the future, including:In addition to the more day-to-day aspects of being on an editorial board, the ECR would also be mentored by the Associate Editors in how to act as an ambassador for the journal. This could be:Over 70 applications were received to the pilot from 17 countries, from Germany to Malaysia and Cyprus to South Africa. Successful ECRs were matched based on their scientific interests to one of the Associate Editors who agreed to take part in the pilot from the Biochemical Journal, Biochemical Society Transactions and Clinical Science. The scheme officially began on 1 March 2021. Portland Press is currently monitoring the participants in the pilot scheme and are excited to see how it progresses.Essays in Biochemistry publishes themed issues looking at key topics of interest within the molecular biosciences. The most recent issue, Biochemistry: One Molecule at a Time, explores the emerging area of single-molecule biochemistry, which allows researchers to investigate one biological macromolecule at a time.This issue of Essays in Biochemistry was guest-edited by Dominika and is the very first written entirely by ECRs, highlighting the strength, diversity and excellence of the early career single-molecule scientists who are driving this exciting field forward.The issue includes contributions from Dr Lisanne Spenkelink (post-doc at the University of Wollongong, Australia), Dr Sonja Schmid (post-doc at the Kavli Institute of Nanoscience Delft, The Netherlands), George Cameron (a PhD student at The Francis Crick Institute, UK), Rebecca Andrews (a PhD student at the University of Oxford, UK) and Dr Erik Holmstrom (a junior PI at the University of Kansas, USA).In 2021, the Biochemical Society and Portland Press collaborated to launch our Biochemistry Focus webinar programme. As part of this, we included a series of webinars aimed specifically at ECRs. The series has two strands: a research strand covering a topic of interest submitted by Early Career Members of the Society, and includes presentations from four ECRs who share their current work with the audience, and a strand offering career support and guidance. The Society aims to include one ECR-focused webinar a month, alternating between these strands where possible.The events are available online for free and generally last about an hour in the early afternoon (GMT).Recently covered scientific topics include ‘Novel advances in signalling: next generation approaches’ and ‘Computational biology and bioinformatics’; all past ECR webinars can be viewed on the Society YouTube channel.We have an exciting line-up of ECR webinars over the course of 2021, both research- and career-focused, on topics such as ‘glycobiology’ and ‘raising one’s professional profile’.Having conducted two surveys last year to establish the impact of COVID-19 on our community of researchers, the Biochemical Society noted the disproportional effect the pandemic had on ECRs. As with the first survey (which ran in April 2020), a follow-up article in The Biochemist encapsulates the insights garnered by our second survey (conducted October 2020), including:The ECAP was instrumental in formulating and implementing a Society-wide action plan, which aims to mitigate the dramatic impact of the pandemic on the junior members of the bioscience community. Our efforts to support the ECR community are focused particularly on tackling the concerns around career progression and funding landscape; please see our latest policy article in The Biochemist for further details.If you are an ECR, our Early Career Membership brings all the benefits of Full Membership at a significantly reduced cost!Applicants are entitled to Early Career Membership for 10 years from the date of gaining a postgraduate qualification in the life sciences.Find out more about how you can join the Biochemical Society here https://biochemistry.org/membership/early-careers/

In 2020, the Biochemical Society conducted two surveys to assess the impact of the COVID-19 pandemic on researchers in the molecular biosciences. Totalling over 1000 responses across both surveys (a first-phase survey launched in April 2020 and a follow-up survey launched in October 2020) , the feedback reflected the impact of the various lockdowns and restrictions implemented within both the UK and globally.

Biological processes are orchestrated by complex networks of molecules. Conventional approaches for studying the action of biomolecules operate on a population level, averaging out any inhomogeneities within the ensemble. Investigating one biological macromolecule at a time allows researchers to directly probe individual behaviours, and thus characterise the intrinsic molecular heterogeneity of the system. Single-molecule methods have unravelled unexpected modes of action for many seemingly well-characterised biomolecules and often proved instrumental in understanding the intricate mechanistic basis of biological processes. This collection of reviews aims to showcase how single-molecule techniques can be used to address important biological questions and to inspire biochemists to 'zoom in' to the population and probe individual molecular behaviours, beyond the ensemble average. Furthermore, this issue of Essays in Biochemistry is the very first written and edited entirely by early career researchers, and so it also highlights the strength, diversity and excellence of the younger generation single-molecule scientists who drive this exciting field of research forward.

The COVID-19 pandemic has affected scientific research across the world, emphasizing old entrenched problems as well as bringing new challenges and even some opportunities. In May 2020, the Biochemical Society conducted a survey of 469 researchers, across all career stages, to assess the impact of the pandemic on the molecular bioscience community. The survey results indicated that researchers early in their careers are the most adversely affected by the ongoing circumstances.

SUMMARYFaithful replication of chromatin domains during cell division is fundamental to eukaryotic development. During replication, nucleosomes are disrupted ahead of the replication fork, followed by their rapid reassembly on daughter strands from the pool of recycled parental and newly synthesized histones. Here, we use single-molecule imaging and replication assays in Xenopus laevis egg extracts to determine the outcome of replication fork encounters with nucleosomes. Contrary to current models, the majority of parental histones are evicted from the DNA, with histone recycling, nucleosome sliding and replication fork stalling also occurring but at lower frequencies. The anticipated local histone transfer only becomes dominant upon depletion of free histones from extracts. Our studies provide the first direct evidence that parental histones remain in close proximity to their original locus during recycling and reveal that provision of excess histones results in impaired histone recycling, which has the potential to affect epigenetic memory.

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Many human proteins contain intrinsically disordered regions, and disorder in these proteins can be fundamental to their function-for example, facilitating transient but specific binding, promoting allostery, or allowing efficient posttranslational modification. SasG, a multidomain protein implicated in host colonization and biofilm formation in Staphylococcus aureus, provides another example of how disorder can play an important role. Approximately one-half of the domains in the extracellular repetitive region of SasG are intrinsically unfolded in isolation, but these E domains fold in the context of their neighboring folded G5 domains. We have previously shown that the intrinsic disorder of the E domains mediates long-range cooperativity between nonneighboring G5 domains, allowing SasG to form a long, rod-like, mechanically strong structure. Here, we show that the disorder of the E domains coupled with the remarkable stability of the interdomain interface result in cooperative folding kinetics across long distances. Formation of a small structural nucleus at one end of the molecule results in rapid structure formation over a distance of 10 nm, which is likely to be important for the maintenance of the structural integrity of SasG. Moreover, if this normal folding nucleus is disrupted by mutation, the interdomain interface is sufficiently stable to drive the folding of adjacent E and G5 domains along a parallel folding pathway, thus maintaining cooperative folding.

Bacteria exploit surface proteins to adhere to other bacteria, surfaces and host cells. Such proteins need to project away from the bacterial surface and resist significant mechanical forces. SasG is a protein that forms extended fibrils on the surface of Staphylococcus aureus and promotes host adherence and biofilm formation. Here we show that although monomeric and lacking covalent cross-links, SasG maintains a highly extended conformation in solution. This extension is mediated through obligate folding cooperativity of the intrinsically disordered E domains that couple non-adjacent G5 domains thermodynamically, forming interfaces that are more stable than the domains themselves. Thus, counterintuitively, the elongation of the protein appears to be dependent on the inherent instability of its domains. The remarkable mechanical strength of SasG arises from tandemly arrayed ‘clamp’ motifs within the folded domains. Our findings reveal an elegant minimal solution for the assembly of monomeric mechano-resistant tethers of variable length.

SasG has a long repeat region made up of identical repeating E and G5 domains. Although the domains themselves are relatively unstable (indeed E domains on their own are unfolded), the cooperative folding of the domains results in formation of molecules that are long and remarkably mechanically resistant. We have used small angle X-ray scattering and mechanical unfolding methods, combined with simulations, to show that SasG constructs of physiological length are indeed monomeric, highly extended and mechanically strong. Obligate folding cooperativity of the intrinsically disordered E domain couples spatially separate G5 domains both thermodynamically and structurally, creating a superstructure that supersedes the domain architecture. Our findings provide a simple solution for the efficient assembly of mechano-resistant elongated structures of tunable length from a single polypeptide chain and have significant potential for the development of novel biomaterials.

Although there have been many thermodynamic studies of the association of intrinsically disordered proteins (IDPs) with their binding partners, there have been relatively few kinetic studies. We have analysed the association of two different IDP systems, which form helical structure upon binding. These systems have markedly different behaviour. One associates very slowly and weakly, and the other very rapidly and strongly. The kinetic data therefore need to be analysed in a different manner to each other, and to the folding of single domain proteins. We discuss why standard approaches developed for folded protein-protein interactions may not be appropriate for reactions where one or both proteins are disordered. In addition, we show how protein engineering can be used to provide insights into the pathways/mechanisms of folding. Finally we have analysed the kinetics of the folding of an intrinsically disordered region (IDR) within a protein - a domain that only folds when attached to a neighbouring folded domain. This multidomain protein folds in a very different manner compared to those formed from adjacent domains which can fold in isolation.

The authors regret that there is an typographical error in the DNA sequence shown in Fig. 1b and the graphical abstract of this paper: The G and C bases of the right-hand tetraloop as drawn (positions 32 and 35) should be transposed; the correct sequence of this loop is ‑C32-T33-T34-G35-. Please note that the sequences shown in Supplementary Table 3 do not suffer this error, and are correct. Also, in Table 1 of the main paper the average atomic rms deviation from the average structure for the backbone atoms (N, Ca, C) of PARP-1 finger 1 (residues 7-93) should be 0.48 ± 0.11A (rather than 0.50 ± 0.12A). The corrected Fig. 1 and Graphical Abstract appear on next page. Fig. 1 Graphical Abstract

Staphylococcus aureus and Staphylococcus epidermidis form communities (called biofilms) on inserted medical devices, leading to infections that affect many millions of patients worldwide and cause substantial morbidity and mortality. As biofilms are resistant to antibiotics, device removal is often required to resolve the infection. Thus, there is a need for new therapeutic strategies and molecular data that might assist their development. Surface proteins S. aureus surface protein G (SasG) and accumulation-associated protein (S. epidermidis) promote biofilm formation through their "B" regions. B regions contain tandemly arrayed G5 domains interspersed with approximately 50 residue sequences (herein called E) and have been proposed to mediate intercellular accumulation through Zn2+-mediated homodimerization. Although E regions are predicted to be unstructured, SasG and accumulation-associated protein form extended fibrils on the bacterial surface. Here we report structures of E-G5 and G5-E-G5 from SasG and biophysical characteristics of single and multidomain fragments. E sequences fold cooperatively and form interlocking interfaces with G5 domains in a head-to-tail fashion, resulting in a contiguous, elongated, monomeric structure. E and G5 domains lack a compact hydrophobic core, and yet G5 domain and multidomain constructs have thermodynamic stabilities only slightly lower than globular proteins of similar size. Zn2+ does not cause SasG domains to form dimers. The work reveals a paradigm for formation of fibrils on the 100-nm scale and suggests that biofilm accumulation occurs through a mechanism distinct from the "zinc zipper." Finally, formation of two domains by each repeat (as in SasG) might reduce misfolding in proteins when the tandem arrangement of highly similar sequences is advantageous.

Poly(ADP-ribose)polymerase-1 (PARP-1) is a highly abundant chromatin-associated enzyme present in all higher eukaryotic cell nuclei, where it plays key roles in the maintenance of genomic integrity, chromatin remodeling and transcriptional control. It binds to DNA single- and double-strand breaks through an N-terminal region containing two zinc fingers, F1 and F2, following which its C-terminal catalytic domain becomes activated via an unknown mechanism, causing formation and addition of polyadenosine-ribose (PAR) to acceptor proteins including PARP-1 itself. Here, we report a biophysical and structural characterization of the F1 and F2 fingers of human PARP-1, both as independent fragments and in the context of the 24-kDa DNA-binding domain (F1 + F2). We show that the fingers are structurally independent in the absence of DNA and share a highly similar structural fold and dynamics. The F1 + F2 fragment recognizes DNA single-strand breaks as a monomer and in a single orientation. Using a combination of NMR spectroscopy and other biophysical techniques, we show that recognition is primarily achieved by F2, which binds the DNA in an essentially identical manner whether present in isolation or in the two-finger fragment. F2 interacts much more strongly with nicked or gapped DNA ligands than does F1, and we present a mutational study that suggests origins of this difference. Our data suggest that different DNA lesions are recognized by the DNA-binding domain of PARP-1 in a highly similar conformation, helping to rationalize how the full-length protein participates in multiple steps of DNA single-strand breakage and base excision repair.

The SasG surface protein of Staphylococcus aureus has been shown to promote the formation of biofilm. SasG comprises an N-terminal A domain and repeated B domains. Here we demonstrate that SasG is involved in the accumulation phase of biofilm, a process that requires a physiological concentration of Zn(2+). The B domains, but not the A domain, are required. Purified recombinant B domain protein can form dimers in vitro in a Zn(2+)-dependent fashion. Furthermore, the protein can bind to cells that have B domains anchored to their surface and block biofilm formation. The full-length SasG protein exposed on the cell surface is processed within the B domains to a limited degree, resulting in cleaved proteins of various lengths being released into the supernatant. Some of the released molecules associate with the surface-exposed B domains that remain attached to the cell. Studies using inhibitors and mutants failed to identify any protease that could cause the observed cleavage within the B domains. Extensively purified recombinant B domain protein is very labile, and we propose that cleavage occurs spontaneously at labile peptide bonds and that this is necessary for biofilm formation.