Escherichia coli DNA repair helicase Lhr is also a uracil-DNA glycosylase.

DNA glycosylases protect genetic fidelity during DNA replication by removing potentially mutagenic chemically damaged DNA bases. Bacterial Lhr proteins are well-characterized DNA repair helicases that are fused to additional 600-700 amino acids of unknown function, but with structural homology to SecB chaperones and AlkZ DNA glycosylases. Here, we identify that Escherichia coli Lhr is a uracil-DNA glycosylase (UDG) that depends on an active site aspartic acid residue. We show that the Lhr DNA helicase activity is functionally independent of the UDG activity, but that the helicase domains are required for fully active UDG activity. Consistent with UDG activity, deletion of lhr from the E. coli chromosome sensitized cells to oxidative stress that triggers cytosine deamination to uracil. The ability of Lhr to translocate single-stranded DNA and remove uracil bases suggests a surveillance role to seek and remove potentially mutagenic base changes during replication stress.

Advancing structural biology through breakthroughs in AI.

The past century has witnessed an exponential increase in our atomic-level understanding of molecular and cellular mechanisms from a structural perspective, with multiple landmark achievements contributing to the field. This, coupled with recent and continuing breakthroughs in artificial intelligence methods such as AlphaFold2, and enhanced computational power, is enabling our understanding of protein structure and function at unprecedented levels of accuracy and predictivity. Here, we describe some of the major recent advances across these fields, and describe, as these technologies coalesce, the potential to utilise our enhanced knowledge of intricate cellular and molecular systems to discover novel therapeutics to alleviate human suffering.

The HelQ human DNA repair helicase utilizes a PWI-like domain for DNA loading through interaction with RPA, triggering DNA unwinding by the HelQ helicase core.

Genome instability is a characteristic enabling factor for carcinogenesis. HelQ helicase is a component of human DNA maintenance systems that prevent or reverse genome instability arising during DNA replication. Here, we provide details of the molecular mechanisms that underpin HelQ function-its recruitment onto ssDNA through interaction with replication protein A (RPA), and subsequent translocation of HelQ along ssDNA. We describe for the first time a functional role for the non-catalytic N-terminal region of HelQ, by identifying and characterizing its PWI-like domain. We present evidence that this domain of HelQ mediates interaction with RPA that orchestrates loading of the helicase domains onto ssDNA. Once HelQ is loaded onto the ssDNA, ATP-Mg2+ binding in the catalytic site activates the helicase core and triggers translocation along ssDNA as a dimer. Furthermore, we identify HelQ-ssDNA interactions that are critical for the translocation mechanism. Our data are novel and detailed insights into the mechanisms of HelQ function relevant for understanding how human cells avoid genome instability provoking cancers, and also how cells can gain resistance to treatments that rely on DNA crosslinking agents.

A new chapter for a better Bioscience Reports.

As Bioscience Reports enters its fifth decade of continuous multidisciplinary life science publishing, here we present a timely overview of the journal. In addition to introducing ourselves and new Associate Editors for 2021, we reflect on the challenges the new Editorial Board has faced and overcome since we took over the editorial leadership in June of 2020, and detail some key strategies on how we plan to encourage more submissions and broader readership for a better and stronger journal in the coming years.

Crystal structure and molecular dynamics of human POLDIP2, a multifaceted adaptor protein in metabolism and genome stability.

Polymerase δ-interacting protein 2 (POLDIP2, PDIP38) is a multifaceted, "moonlighting" protein, involved in binding protein partners from many different cellular processes, including mitochondrial metabolism and DNA replication and repair. How POLDIP2 interacts with many different proteins is unknown. Towards this goal, we present the crystal structure of POLDIP2 to 2.8 Å, which exhibited a compact two-domain ß-strand-rich globular structure, confirmed by circular dichroism and small angle X-ray scattering approaches. POLDIP2 comprised canonical DUF525 and YccV domains, but with a conserved domain linker packed tightly, resulting in an "extended" YccV module. A central channel was observed, which we hypothesize could influence structural changes potentially mediated by redox conditions, following observation of a modified cysteine residue in the channel. Unstructured regions were rebuilt by ab initio modelling to generate a model of full-length POLDIP2. Molecular dynamics simulations revealed a highly dynamic N-terminal region tethered to the YccV-domain by an extended linker, potentially facilitating interactions with distal binding partners. Models of POLDIP2 complexed with two of its partners, PrimPol and PCNA, indicated that dynamic flexibility of the POLDIP2 N-terminus and loop regions likely mediate protein interactions.

Mechanistic insights into Lhr helicase function in DNA repair.

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the 'parental' DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication.

The Controversial Roles of ADP-Ribosyl Hydrolases MACROD1, MACROD2 and TARG1 in Carcinogenesis.

Post-translational modifications (PTM) of proteins are crucial for fine-tuning a cell's response to both intracellular and extracellular cues. ADP-ribosylation is a PTM, which occurs in two flavours: modification of a target with multiple ADP-ribose moieties (poly(ADP-ribosyl)ation or PARylation) or with only one unit (MARylation), which are added by the different enzymes of the PARP family (also known as the ARTD family). PARylation has been relatively well-studied, particularly in the DNA damage response. This has resulted in the development of PARP inhibitors such as olaparib, which are increasingly employed in cancer chemotherapeutic approaches. Despite the fact that the majority of PARP enzymes catalyse MARylation, MARylation is not as well understood as PARylation. MARylation is a dynamic process: the enzymes reversing intracellular MARylation of acidic amino acids (MACROD1, MACROD2, and TARG1) were discovered in 2013. Since then, however, little information has been published about their physiological function. MACROD1, MACROD2, and TARG1 have a 'macrodomain' harbouring the catalytic site, but no other domains have been identified. Despite the lack of information regarding their cellular roles, there are a number of studies linking them to cancer. However, some of these publications oppose each other, some rely on poorly-characterised antibodies, or on aberrant localisation of overexpressed rather than native protein. In this review, we critically assess the available literature on a role for the hydrolases in cancer and find that, currently, there is limited evidence for a role for MACROD1, MACROD2, or TARG1 in tumorigenesis.

MicroRNA Quantitation During Dendritic Cell Endocytosis Using Imaging Flow Cytometry: Key Factors and Requirements.

BACKGROUND/AIMS: MicroRNA (miRNA)-induced suppression of dendritic cells (DCs) has been implicated in many diseases. Therefore, accurate monitoring of miRNA endocytosis by DCs is important for understanding the role of miRNAs in many diseases. Recently, a method for measuring the co-localization of Argonaute 2 (AGO2)-associated miRNAs on laser-scanning confocal microscopy method was proposed to localize the miRNAs. But its definition was limited by the number of observed cells through its accuracy. METHODS: In this study, a method based on imaging flow cytometry was developed to localize miR-590-5p with fluorescent probes in DCs. miR-590-5p proven to play an important role in tumor immunity. This method enabled the quantification, visualization and localization of the fluorescence intensity in 30,000 individual cells. RESULTS: Using this method, the DCs with different endocytotic ability were distinguished. The behaviour of miR-590-5p during endocytosis under the stimulation of tumor antigen in DCs was observed, binding to its cognate target mRNA and degradation in DCs. CONCLUSION: This method based on imaging flow cytometry provide an additional method to study miRNA processing in DCs, which makes it a valuable addition to existing miRNA research techniques.

Archaeal DNA polymerases: new frontiers in DNA replication and repair.

Archaeal DNA polymerases have long been studied due to their superior properties for DNA amplification in the polymerase chain reaction and DNA sequencing technologies. However, a full comprehension of their functions, recruitment and regulation as part of the replisome during genome replication and DNA repair lags behind well-established bacterial and eukaryotic model systems. The archaea are evolutionarily very broad, but many studies in the major model systems of both Crenarchaeota and Euryarchaeota are starting to yield significant increases in understanding of the functions of DNA polymerases in the respective phyla. Recent advances in biochemical approaches and in archaeal genetic models allowing knockout and epitope tagging have led to significant increases in our understanding, including DNA polymerase roles in Okazaki fragment maturation on the lagging strand, towards reconstitution of the replisome itself. Furthermore, poorly characterised DNA polymerase paralogues are finding roles in DNA repair and CRISPR immunity. This review attempts to provide a current update on the roles of archaeal DNA polymerases in both DNA replication and repair, addressing significant questions that remain for this field.

Structural insights into the function of ZRANB3 in replication stress response.

Strategies to resolve replication blocks are critical for the maintenance of genome stability. Among the factors implicated in the replication stress response is the ATP-dependent endonuclease ZRANB3. Here, we present the structure of the ZRANB3 HNH (His-Asn-His) endonuclease domain and provide a detailed analysis of its activity. We further define PCNA as a key regulator of ZRANB3 function, which recruits ZRANB3 to stalled replication forks and stimulates its endonuclease activity. Finally, we present the co-crystal structures of PCNA with two specific motifs in ZRANB3: the PIP box and the APIM motif. Our data provide important structural insights into the PCNA-APIM interaction, and reveal unexpected similarities between the PIP box and the APIM motif. We propose that PCNA and ATP-dependency serve as a multi-layered regulatory mechanism that modulates ZRANB3 activity at replication forks. Importantly, our findings allow us to interpret the functional significance of cancer associated ZRANB3 mutations.

N- and C-Terminal Truncations to Enhance Protein Solubility and Crystallization: Predicting Protein Domain Boundaries with Bioinformatics Tools.

Soluble protein expression is a key requirement for biochemical and structural biology approaches to study biological systems in vitro. Production of sufficient quantities may not always be achievable if proteins are poorly soluble which is frequently determined by physico-chemical parameters such as intrinsic disorder. It is well known that discrete protein domains often have a greater likelihood of high-level soluble expression and crystallizability. Determination of such protein domain boundaries can be challenging for novel proteins. Here, we outline the application of bioinformatics tools to facilitate the prediction of potential protein domain boundaries, which can then be used in designing expression construct boundaries for parallelized screening in a range of heterologous expression systems.

Structures of Two Melanoma-Associated Antigens Suggest Allosteric Regulation of Effector Binding.

The MAGE (melanoma associated antigen) protein family are tumour-associated proteins normally present only in reproductive tissues such as germ cells of the testis. The human genome encodes over 60 MAGE genes of which one class (containing MAGE-A3 and MAGE-A4) are exclusively expressed in tumours, making them an attractive target for the development of targeted and immunotherapeutic cancer treatments. Some MAGE proteins are thought to play an active role in driving cancer, modulating the activity of E3 ubiquitin ligases on targets related to apoptosis. Here we determined the crystal structures of MAGE-A3 and MAGE-A4. Both proteins crystallized with a terminal peptide bound in a deep cleft between two tandem-arranged winged helix domains. MAGE-A3 (but not MAGE-A4), is predominantly dimeric in solution. Comparison of MAGE-A3 and MAGE-A3 with a structure of an effector-bound MAGE-G1 suggests that a major conformational rearrangement is required for binding, and that this conformational plasticity may be targeted by allosteric binders.

Structure of the Helicase Domain of DNA Polymerase Theta Reveals a Possible Role in the Microhomology-Mediated End-Joining Pathway.

DNA polymerase theta (Polθ) has been identified as a crucial alternative non-homologous end-joining factor in mammalian cells. Polθ is upregulated in a range of cancer cell types defective in homologous recombination, and knockdown has been shown to inhibit cell survival in a subset of these, making it an attractive target for cancer treatment. We present crystal structures of the helicase domain of human Polθ in the presence and absence of bound nucleotides, and a characterization of its DNA-binding and DNA-stimulated ATPase activities. Comparisons with related helicases from the Hel308 family identify several unique features. Polθ exists as a tetramer both in the crystals and in solution. We propose a model for DNA binding to the Polθ helicase domain in the context of the Polθ tetramer, which suggests a role for the helicase domain in strand annealing of DNA templates for subsequent processing by the polymerase domain.

Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: DETERMINANTS OF DNA BINDING AND REDOX REGULATION BY DISULFIDE BOND FORMATION.

Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors.

Structural insights into the autoregulation and cooperativity of the human transcription factor Ets-2.

Ets-2, like its closely related homologue Ets-1, is a member of the Ets family of DNA binding transcription factors. Both proteins are subject to multiple levels of regulation of their DNA binding and transactivation properties. One such regulatory mechanism is the presence of an autoinhibitory module, which in Ets-1 allosterically inhibits the DNA binding activity. This inhibition can be relieved by interaction with protein partners or cooperative binding to closely separated Ets binding sites in a palindromic arrangement. In this study we describe the 2.5 Å resolution crystal structure of a DNA complex of the Ets-2 Ets domain. The Ets domain crystallized with two distinct species in the asymmetric unit, which closely resemble the autoinhibited and DNA bound forms of Ets-1. This discovery prompted us to re-evaluate the current model for the autoinhibitory mechanism and the structural basis for cooperative DNA binding. In contrast to Ets-1, in which the autoinhibition is caused by a combination of allosteric and steric mechanisms, we were unable to find clear evidence for the allosteric mechanism in Ets-2. We also demonstrated two possibly distinct types of cooperative binding to substrates with Ets binding motifs separated by four and six base pairs and suggest possible molecular mechanisms for this behavior.

Recent advances in the structural molecular biology of Ets transcription factors: interactions, interfaces and inhibition.

The Ets family of eukaryotic transcription factors is based around the conserved Ets DNA-binding domain. Although their DNA-binding selectivity is biochemically and structurally well characterized, structures of homodimeric and ternary complexes point to Ets domains functioning as versatile protein-interaction modules. In the present paper, we review the progress made over the last decade to elucidate the structural mechanisms involved in modulation of DNA binding and protein partner selection during dimerization. We see that Ets domains, although conserved around a core architecture, have evolved to utilize a variety of interaction surfaces and binding mechanisms, reflecting Ets domains as dynamic interfaces for both DNA and protein interaction. Furthermore, we discuss recent advances in drug development for inhibition of Ets factors, and the roles structural biology can play in their future.

Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases.

Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein "3-box" motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.

Mutations in multidomain protein MEGF8 identify a Carpenter syndrome subtype associated with defective lateralization.

Carpenter syndrome is an autosomal-recessive multiple-congenital-malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor-like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. We did not observe any subject with biallelic loss-of function mutations, suggesting that some residual MEGF8 function might be necessary for survival and might influence the phenotypes observed.

Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation.

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome.