Genomic integrity is constantly threatened by problems encountered by the replication fork. BRCA1, BRCA2 and a subset of Fanconi Anaemia proteins protect stalled replication forks from nuclease degradation through pathways involving RAD51. The contribution and regulation of BRCA1 in replication fork protection, and whether this relates to, or differs from, BRCA1's role in homologous recombination (HR) is not clear. Here we show that the canonical BRCA1-PALB2 interaction is not required for fork protection but instead BRCA1-BARD1 is regulated through a conformational change mediated by the phosphorylation-directed prolyl isomerase, PIN1. PIN1 activity enhances BRCA1-BARD1 interaction with RAD51 and consequently RAD51's presence at stalled replication structures. We identify patient missense variants in the regulated BRCA1-BARD1 regions which show poor nascent strand protection but remain proficient for HR, defining novel domains required for fork protection associated with cancer development. Together these findings reveal a previously unrecognised pathway that governs BRCA1-mediated replication fork protection. Main Text Fork progression can be slowed by conflicts with transcription, deoxyribonucleotide (dNTP) shortage or by difficult to replicate sequences, frequently causing fork stalling 1. In order to prevent stalled forks collapsing into DNA double strand breaks (DSBs), a number of responses are elicited including fork remodelling and subsequent nascent strand protection. Agents that cause replicative stress or compromise DNA Polymerase-α function result in a proportion of forks reversing (reviewed in 2,3). The regressed arm of nascent DNA in reversed forks resembles a single-ended DNA DSB which is protected from excessive resection by RAD51. Several factors contribute to RAD51-mediated fork protection including BRCA1/2, FANCA/D2, RAD51 paralogs, BOD1L, SETD1A, WRNIP and Abro1 2 .

The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme.

Homologous recombination (HR) is a pathway to faithfully repair DNA double-strand breaks (DSBs). At the core of this pathway is a DNA recombinase, which, as a nucleoprotein filament on ssDNA, pairs with homologous DNA as a template to repair the damaged site. In eukaryotes Rad51 is the recombinase capable of carrying out essential steps including strand invasion, homology search on the sister chromatid and strand exchange. Importantly, a tightly regulated process involving many protein factors has evolved to ensure proper localisation of this DNA repair machinery and its correct timing within the cell cycle. Dysregulation of any of the proteins involved can result in unchecked DNA damage, leading to uncontrolled cell division and cancer. Indeed, many are tumour suppressors and are key targets in the development of new cancer therapies. Over the past 40 years, our structural and mechanistic understanding of homologous recombination has steadily increased with notable recent advancements due to the advances in single particle cryo electron microscopy. These have resulted in higher resolution structural models of the signalling proteins ATM (ataxia telangiectasia mutated), and ATR (ataxia telangiectasia and Rad3-related protein), along with various structures of Rad51. However, structural information of the other major players involved, such as BRCA1 (breast cancer type 1 susceptibility protein) and BRCA2 (breast cancer type 2 susceptibility protein), has been limited to crystal structures of isolated domains and low-resolution electron microscopy reconstructions of the full-length proteins. Here we summarise the current structural understanding of homologous recombination, focusing on key proteins in recruitment and signalling events as well as the mediators for the Rad51 recombinase. Keywords Homologous recombination • Cryo electron microscopy • X-ray crystallography • Double-strand break repair • DNA damage signalling and repair Abbreviations ADP Adenosine diphosphate AMP-PNP Adenylyl-imidodiphosphate ATM Ataxia telangiectasia mutated ATP Adenosine triphosphate ATR Ataxia telangiectasia and Rad3-related protein BARD1 BRCA1-associated RING domain protein 1 BRCA1 Breast cancer type 1 susceptibility protein BRCA2 Breast cancer type 2 susceptibility protein BRCT BRCA1 C-terminal domain cryoEM Cryo electron microscopy DDR DNA damage response DSB Double-strand break EJ End joining MRE11 Meiotic recombination 11 homolog 1 MRN MRE11 RAD50 NBS1 NBS1 Nijmegen breakage syndrome protein 1 OB Oligonucleotide/oligosaccharide-binding PALB2 Partner and localiser of BRCA2 RPA Replication protein A

Classic galactosemia is a potentially lethal disease caused by the dysfunction of galactose 1-phosphate uridylyltransferase (GALT). Over 300 disease-associated GALT mutations have been reported, with the majority being missense changes, although a better understanding of their underlying molecular effects has been hindered by the lack of structural information for the human enzyme. Here, we present the 1.9 Å resolution crystal structure of human GALT (hGALT) ternary complex, revealing a homodimer arrangement that contains a covalent uridylylated intermediate and glucose-1-phosphate in the active site, as well as a structural zinc-binding site, per monomer. hGALT reveals significant structural differences from bacterial GALT homologues in metal ligation and dimer interactions, and therefore is a zbetter model for understanding the molecular consequences of disease mutations. Both uridylylation and zinc binding influence the stability and aggregation tendency of hGALT. This has implications for disease-associated variants where p.Gln188Arg, the most commonly detected, increases the rate of aggregation in the absence of zinc likely due to its reduced ability to form the uridylylated intermediate. As such our structure serves as a template in the future design of pharmacological chaperone therapies and opens new concepts about the roles of metal binding and activity in protein misfolding by disease-associated mutants.

Glycogen branching enzyme 1 (GBE1) plays an essential role in glycogen biosynthesis by generating α-1,6-glucosidic branches from α-1,4-linked glucose chains, to increase solubility of the glycogen polymer. Mutations in the GBE1 gene lead to the heterogeneous early-onset glycogen storage disorder type IV (GSDIV) or the late-onset adult polyglucosan body disease (APBD). To better understand this essential enzyme, we crystallized human GBE1 in the apo form, and in complex with a tetra- or hepta-saccharide. The GBE1 structure reveals a conserved amylase core that houses the active centre for the branching reaction and harbours almost all GSDIV and APBD mutations. A non-catalytic binding cleft, proximal to the site of the common APBD mutation p.Y329S, was found to bind the tetra- and hepta-saccharides and may represent a higher-affinity site employed to anchor the complex glycogen substrate for the branching reaction. Expression of recombinant GBE1-p.Y329S resulted in drastically reduced protein yield and solubility compared with wild type, suggesting this disease allele causes protein misfolding and may be amenable to small molecule stabilization. To explore this, we generated a structural model of GBE1-p.Y329S and designed peptides ab initio to stabilize the mutation. As proof-of-principle, we evaluated treatment of one tetra-peptide, Leu-Thr-Lys-Glu, in APBD patient cells. We demonstrate intracellular transport of this peptide, its binding and stabilization of GBE1-p.Y329S, and 2-fold increased mutant enzymatic activity compared with untreated patient cells. Together, our data provide the rationale and starting point for the screening of small molecule chaperones, which could become novel therapies for this disease.

Background: Human cystathionine β-synthase (hCBS) is activated by S-adenosyl-l-methionine (AdoMet).Results: We presented structural and solution evidence that AdoMet binding to hCBS regulatory domain causes conformational rearrangement to the protein.Conclusion: AdoMet activates hCBS by altering the interface and arrangement between its catalytic and regulatory domains.Significance: Our data identified the key residues for AdoMet binding and provide a mechanism for allosteric activation.

Type I galactosemia is a genetic disorder that is caused by the impairment of galactose-1-phosphate uridylyltransferase (GALT; EC 2.7.7.12). Although a large number of mutations have been detected through genetic screening of the human GALT (hGALT) locus, for many it is not known how they cause their effects. The majority of these mutations are missense, with predicted substitutions scattered throughout the enzyme structure and thus causing impairment by other means rather than direct alterations to the active site. To clarify the fundamental, molecular basis of hGALT impairment we studied five disease-associated variants p.D28Y, p.L74P, p.F171S, p.F194L and p.R333G using both a yeast model and purified, recombinant proteins. In a yeast expression system there was a correlation between lysate activity and the ability to rescue growth in the presence of galactose, except for p.R333G. Kinetic analysis of the purified proteins quantified each variant’s level of enzymatic impairment and demonstrated that this was largely due to altered substrate binding. Increased surface hydrophobicity, altered thermal stability and changes in proteolytic sensitivity were also detected. Our results demonstrate that hGALT requires a level of flexibility to function optimally and that altered folding is the underlying reason of impairment in all the variants tested here. This indicates that misfolding is a common, molecular basis of hGALT deficiency and suggests the potential of pharmacological chaperones and proteostasis regulators as novel therapeutic approaches for type I galactosemia.

Background: Two intracellular proteins, MMACHC and MMADHC, functionally interact for cobalamin trafficking.Results: MMADHC crystal structure reveals protein-interacting regions and unexpected homology to MMACHC; mutations on either protein interfere with complex formation via different mechanisms.Conclusion: Complex formation likely depends on prior cobalamin processing and can be broken by disease mutations.Significance: MMACHC-MMADHC heterodimerization forms the essential trafficking chaperone delivering cobalamin to client enzymes.