Dr Christopher Cooper

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 E. coli Lhr is a uracil-DNA glycosylase that depends on an active site aspartic acid residue. We show that the Lhr DNA helicase activity is functionally independent of the uracil-DNA glycosylase activity, but that the helicase domains are required for fully active uracil DNA glycosylase activity. Consistent with uracil DNA glycosylase 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.

Producing purified human proteins with high yield and purity remains a considerable challenge. We describe the methods utilized in the Structural Genomics Consortium (SGC) in Oxford, resulting in successful purification of 48% of human proteins attempted; of those, the structures of similar to 40% were solved by X-ray crystallography. The main driver has been the parallel processing of multiple (typically 9-20) truncated constructs of each target; modest diversity in vectors and host systems; and standardized purification procedures. We provide method details as well as data on the properties of the constructs leading to crystallized proteins and the impact of methodological variants. These can be used to formulate guidelines for initial approaches to expression of new eukaryotic proteins. (C) 2010 Elsevier Inc. All rights reserved.

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.

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.

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.

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.

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 β‐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. PDB Code(s): 6Z9C;

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.

Crystallographic analysis of the catalytic domain of PHD finger protein 8 (PHF8), an N-epsilon-methyl lysine histone demethylase associated with mental retardation and cleft lip/palate, reveals a double-stranded beta-helix fold with conserved Fe(II) and cosubstrate binding sites typical of the 2-oxoglutarate dependent oxygenases. The PHF8 active site is highly conserved with those of the FBXL10/11demethylases, which are also selective for the di-/mono-methylated lysine states, but differs from that of the JMJD2 demethylases which are selective for tri-/di-methylated states. The results rationalize the lack of activity for the clinically observed F279S PHF8 variant and they will help to identify inhibitors selective for specific N-epsilon-methyl lysine demethylase subfamilies. (C) 2010 Published by Elsevier B. V. on behalf of the Federation of European Biochemical Societies.

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.

MicroRNAs (miRNA) are a recently discovered class of short non-coding RNA molecules that negatively regulate gene expression. They have been shown to play a critical role in many biological functions. In humans about 320 miRNAs have been identified, some of which are expressed in a cell-specific and developmental stage-specific manner. Recently it has been shown that the expression profile of miRNAs can be used to subtype clinical cases (and cell lines) according to diagnosis with a greater degree of accuracy than traditional gene expression analysis. The identity of miRNAs associated with different lymphoma types however remains poorly defined. Previous expression studies have revealed the presence of at least two subtypes of diffuse large B-cell lymphoma (DLBCL) representing the postulated cell of origin; those that are germinal center B cell derived (GCB-type) and those that are activated B-cell derived (ABC-type). The latter subtype has been linked with poor prognostic outcome. It is not known whether these subtypes are also defined at the miRNA level. Therefore we examined the miRNA expression profile of DLBCL cell lines of defined subtypes as well as sub-populations of B-lymphocytes by microarray analysis. Consistent with recent publications, we found that mir-19a, 19b and 17-5p (part of mir-17-92 cluster) were up-regulated in cell lines but not in normal lymphocyte populations. Furthermore, cluster analysis showed that GCB-type cell lines (SUD-HL4, SUD-HL6 & SUD-HL10) have a distinct miRNA profile from ABC-type cell lines (OCI-Ly3 & OCI-Ly10). Most notably, high levels of expression of mir-155, mir-181b and mir-325 were found in ABC-type cell lines whilst high levels of mir-181a were found in GCB-type cell lines. We looked at expression of mir-155, 181a, 143, 145, 378 and 16 in these cell lines as well as clinical cases of DLBCL by RNase-protection assay. Consistent with the microarray data, we found that mir-155 was expressed in ABC-type cell lines but not GCB-type cell lines whilst the converse was true for mir-181a. Clinical cases showed similar patterns of expression but have still to be sub-typed according to immunohistochemical markers. Although still preliminary, our data suggests that miRNA profiling may be a useful tool in predicting the subtype of DLBCL cases and hence clinical outcome.

Mutations of human PHF8 cluster within its JmjC encoding exons and are linked to mental retardation (MR) and a cleft lip/palate phenotype. Sequence comparisons, employing structural insights, suggest that PHF8 contains the double stranded beta-helix fold and ferrous iron binding residues that are present in 2-oxoglutarate-dependent oxygenases. We report that recombinant PHF8 is an Fe(II) and 2-oxoglutarate-dependent N-epsilon-methyl lysine demethylase, which acts on histone substrates. PHF8 is selective in vitro for N-epsilon-di- and mono-methylated lysine residues and does not accept trimethyl substrates. Clinically observed mutations to the PHF8 gene cluster in exons encoding for the double stranded beta-helix fold and will therefore disrupt catalytic activity. The PHF8 missense mutation c.836C > T is associated with mild MR, mild dysmorphic features, and either unilateral or bilateral cleft lip and cleft palate in two male siblings. This mutant encodes a F279S variant of PHF8 that modifies a conserved hydrophobic region; assays with both peptides and intact histones reveal this variant to be catalytically inactive. The dependence of PHF8 activity on oxygen availability is interesting because the occurrence of fetal cleft lip has been demonstrated to increase with maternal hypoxia in mouse studies. Cleft lip and other congenital anomalies are also linked indirectly to maternal hypoxia in humans, including from maternal smoking and maternal anti-hypertensive treatment. Our results will enable further studies aimed at defining the molecular links between developmental changes in histone methylation status, congenital disorders and MR.

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.

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.

Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers. [Display omitted] •Loss of ALC1 nucleosome remodeling confers PARPi, MMS, and formyl-dU sensitivity•ALC1 is required after lesion excision by DNA glycosylases prior to APEX1•Loss of ALC1 is synthetic lethal with homologous recombination deficiency (HRD)•Endogenous alkylated base damage is a source of synthetic lethality with HRD Hewitt et al. report that loss of the chromatin-remodeling enzyme ALC1 leads to persistent BER intermediates and a critical dependency on HR for repair. Combined loss of ALC1 and HR is synthetic lethal, which can be partially rescued by blocking excision of endogenous alkylation damage by the glycosylase MPG.

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.

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.

Werner syndrome helicase (WRN) is a RecQ-family DNA helicase essential for genome maintenance and is a synthetic lethal target in microsatellite instability-high (MSI-H) cancers. Despite its therapeutic promise, the conformational dynamics that enable WRN to unwind DNA, and how inhibitors disrupt this activity, remains poorly understood. Here, we present crystal structures of apo WRN and WRN bound to single-stranded DNA (ssDNA), capturing key conformations in the helicase catalytic cycle. These structures reveal how WRN engages DNA through conserved polar and aromatic interactions, and how domain rearrangements, including an ordering of the aromatic-rich loop (ARL), drive directional translocation. Biochemical and biophysical data demonstrate how nucleotide and inhibitor binding remodel these conformations and suggest that known clinical inhibitors (HRO761 and VVD-133214) function by locking WRN in inactive, ‘off-DNA’ states. Resistance emerged rapidly in vitro, through acquired point mutations as well as altered WRN expression. Together, our findings provide a structural framework for the WRN structural cycle and support the development of next-generation ‘on-DNA’ inhibitors to overcome resistance. In this study, Kennedy et al. combine crystallography, biophysical measurements and biochemical assays to define a structural cycle for Werner syndrome helicase (WRN) and reveal how nucleotide binding and ssDNA engagement lead to conformational transitions.

The Borrelia genome consists of a linear chromosome and numerous linear and circular plasmids. Using a computational framework, we examined the plasmid proteome of Borrelia burgdorferi sensu lato to identify potential outer membrane (OM) β-barrel proteins. Our approach identified BBJ25 on plasmid lp38 as an Omp85 superfamily domain with structural homology to BamA/TamA. Analysis of the surrounding genes reveal a cluster of seven genes. Structure-function analysis (AlphaFold3 and DALI) points towards a role in membrane transport, with two chaperone proteins (BBJ23, BBJ24), the Omp85-family domain BBJ25, a homodimeric MacB-like type-VII ABC transporter with an associated ATP-binding domain (BBJ26, BBJ27), a LolA-like domain (BBJ28), and one protein of unknown function (BBJ29). Taken together, this points towards a role in the ATP-driven extraction of a non-polar ligand, possibly lipoproteins, from the inner membrane and delivery to the OM. Structural homology is detected to Escherichia coli proteins involved in lipoprotein sorting (LolACDE). Wider analysis of the Borrelia genus revealed two major variants of this 7-gene cluster, distributed across multiple different linear elements including lp17, lp28, lp38, and the linear chromosome of Borrelia turcica. Borrelia valaisiana was found to have both allelic variants coexisting in the same genospecies on different linear plasmids (lp28-3 and lp28-8). Previous studies have reported up-regulation of these genes in response to mammalian signals. The conservation of these proteins throughout the Borrelia genus, coupled with the occurrence of multiple copies in some genospecies, point towards a critical function. The identity of the target ligand remains uncertain and requires experimental verification.