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DOE OSTI2021

Association of somatic mutations in BRCA2 BRC domain with chemotherapy sensitivity and survival in high grade serous ovarian cancer

Highlights: • BRCA2 BRC domain-associated mutation confers higher platinum sensitivity of HGSOC. • Somatic BRCA2 BRC domain mutation impairs BRCA2 HR in HGSOC. • BRCA2 BRC domain mutation at sites is crucial for HR repair of DNA DSB induced by platinums. • Somatic BRCA2 BRC domain missense mutations improve outcomes in patients with HGSOC. Mutations at sites crucial for the interaction between RAD51 and BRC domains impair the ability of BRCA2 homologous recombination. We aimed to clarify whether BRCA2 BRC domain-associated mutation correlates with sensibility of platinum-based chemotherapy and survival in high-grade serous ovarian cancer (HGSOC).

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Build Optimization Software Tools (BOOST) v2.0.0

Build Optimization Software Tools (BOOST) accelerate the design of DNA, RNA and protein sequences for their synthesis and assembly in an automated and scalable fashion. The BOOST Juggler automates the following two design tasks: - Reverse-Translation of protein sequences into DNA sequences - Codon Juggling of DNA sequences The BOOST Polisher provides the following design tasks: - The Verification of DNA sequences against DNA synthesis constraints - The Modification of DNA sequences in case they violate any DNA synthesis constraint In addition, the BOOST Polisher can be instructed to verify and modify DNA sequences against sequence patterns, such as restriction sites. The BOOST Partitioner supports both Gibson/chewback and Yeast assembly (also known as Transformation-Associated Recombination, or TAR) methods when performing the decomposition of large sequences that exceed the maximum length of DNA synthesis into synthesizable building blocks. The Partitioner can be instructed to find building blocks with appropriate overlap sequences, making their assembly into the complete construct more efficient. BOOST Workflows enable, in a customized fashion, the execution of the Juggler, Polisher, and Partitioner tools on a batch of sequences.

Sharkey, Michael↗

DOE OSTI2020

An atypical BRCT–BRCT interaction with the XRCC1 scaffold protein compacts human DNA Ligase IIIα within a flexible DNA repair complex

The XRCC1–DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT–BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS–MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT–BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Identification of a new circulating recombinant form of human immunodeficiency virus type 1, CRF124_cpx involving subtypes A, G, H, and CRF27_cpx in Angola

Angola, located in Central Africa, has around 320,000 (270,000–380,000) people living with human immunodeficiency virus (HIV)/AIDS, equivalent to 1% of the country’s population at the end of 2021. A previous study conducted in 2012, using Angolan samples collected between 2008 and 2010 revealed a high prevalence of HIV-1 recombinants, around 42% of sequences, with 21% showing the same UH profile in partial pol region which were grouped into a monophyletic cluster with high bootstrap support. Thus, the objective of the present work was to obtain complete genomes of those sequences and characterize them, aiming at a description of a new circulating recombinant form (CRF). Whole blood from nine HIV-1 UH pol -infected individuals had their genomic DNA extracted, and nested PCR was used to amplify seven overlapping fragments targeting the full-length HIV-1 genome. The final classification was based on maximum likelihood trees, and recombination analyses were performed using a bootscan from the Simplot program. BLAST and Los Alamos Database inspections were used to search other similar H-like pol sequences. Complete genome amplification was possible for three samples, partial genomes were obtained for the other three, and only pol was available for the remaining three sequences. Bootscan analysis of the two whole-genome and three partial genome sequences retrieved from people living with HIV/AIDS (PLHIVA) without epidemiological linkage showed the same complex recombination profile involving HIV-1 subtypes A/G/H/CRF27_cpx, with a total of six recombinant breakpoints, aiming to classify a new HIV-1 CRF124_cpx. We found no other full-length HIV-1 genomes with the same mosaic profile; however, we identified 33 partial pol sequences, mainly sampled from Angola between 2001 to 2019, with the same H-like profile. Bayesian analysis of H and H-like pol sequences indicates that CRF124_cpx probably originated in Angola at mid-1970s, indicating that this CRF has been circulating in the country for a long time. In summary, our study describes a new CRF circulating principally in Angola and highlights the importance of continuing molecular surveillance studies, especially in countries with high molecular diversity of HIV.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Structure and in vivo psoralen DNA crosslink repair activity of mycobacterial Nei2

Mycobacterium smegmatis Nei2 is a monomeric enzyme with AP β-lyase activity on single-stranded DNA. Expression of Nei2, and its operonic neighbor Lhr (a tetrameric 3'-to-5' helicase), is induced in mycobacteria exposed to DNA damaging agents. Here, we find that nei2 deletion sensitizes M. smegmatis to killing by DNA inter-strand crosslinker trimethylpsoralen but not to crosslinkers mitomycin C and cisplatin. By contrast, deletion of lhr sensitizes to killing by all three crosslinking agents. We report a 1.45 Å crystal structure of recombinant Nei2, which is composed of N and C terminal lobes flanking a central groove suitable for DNA binding. The C lobe includes a tetracysteine zinc complex. Mutational analysis identifies the N-terminal proline residue (Pro2 of the ORF) and Lys51, but not Glu3, as essential for AP lyase activity. We find that Nei2 has 5-hydroxyuracil glycosylase activity on single-stranded DNA that is effaced by alanine mutations of Glu3 and Lys51 but not Pro2. Testing complementation of psoralen sensitivity by expression of wild-type and mutant nei2 alleles in Δnei2 cells established that AP lyase activity is neither sufficient nor essential for crosslink repair. By contrast, complementation of psoralen sensitivity of Δlhr cells by mutant lhr alleles depended on Lhr’s ATPase/helicase activities and its tetrameric quaternary structure. The lhr–nei2 operon comprises a unique bacterial system to rectify inter-strand crosslinks.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Identification, characterization, and structure of a tRNA splicing enzyme RNA 5′-OH kinase from the pathogenic fungi Mucorales

Fungal Trl1 is an essential tRNA splicing enzyme composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that convert the 2′,3′-cyclic-PO 4 and 5′-OH ends of tRNA exons into the 3′-OH,2′-PO 4 and 5′-PO 4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trifunctional Trl1 enzymes are present in most human fungal pathogens and are untapped targets for antifungal drug discovery. Mucorales species, deemed high-priority human pathogens by WHO, elaborate a noncanonical tRNA splicing apparatus in which a stand-alone monofunctional RNA ligase enzyme joins 3′-OH,2′-PO 4 and 5′-PO 4 termini. Here we identify a stand-aloneMucor circinelloidespolynucleotide kinase (MciKIN) and affirm its biological activity in tRNA splicing by genetic complementation in yeast. Recombinant MciKIN catalyzes magnesium-dependent phosphorylation of 5′-OH RNA and DNA ends in vitro. MciKIN displays a strong preference for GTP as the phosphate donor in the kinase reaction, a trait shared with the stand-alone RNA kinase homologs from Mucorales speciesRhizopus azygosporus(RazKIN) andLichtheimia corymbifera(LcoKIN) and with the kinase domains of fungal Trl1 enzymes. We report a 1.65 Å crystal structure of RazKIN in complex with GDP•Mg 2+ that illuminates the basis for guanosine nucleotide specificity.

Biochemistry & Molecular Biology↗

DOE OSTI2020

Determinants for Efficient Editing with Cas9-Mediated Recombineering in Escherichia coli

In E. coli, editing efficiency with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA-mediated DNA double-strand break (DSB)-induced cell death. We found that editing efficiency with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, nontargeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise editing efficiency. These effects on editing efficiency were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs, as well as possible differences in binding of Cas9:gRNA complexes to their target sites, account for the observed variations in editing efficiency between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

ATM signaling modulates cohesin behavior in meiotic prophase and proliferating cells

Cohesins are ancient and ubiquitous regulators of chromosome architecture and function, but their diverse roles and regulation remain poorly understood. During meiosis, chromosomes are reorganized as linear arrays of chromatin loops around a cohesin axis. This unique organization underlies homolog pairing, synapsis, double-stranded break induction, and recombination. We report that axis assembly in Caenorhabditis elegans is promoted by DNA-damage response (DDR) kinases that are activated at meiotic entry, even in the absence of DNA breaks. Downregulation of the cohesin-destabilizing factor WAPL-1 by ATM-1 promotes axis association of cohesins containing the meiotic kleisins COH-3 and COH-4. ECO-1 and PDS-5 also contribute to stabilizing axis-associated meiotic cohesins. Further, our data suggest that cohesin-enriched domains that promote DNA repair in mammalian cells also depend on WAPL inhibition by ATM. Thus, DDR and Wapl seem to play conserved roles in cohesin regulation in meiotic prophase and proliferating cells.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

In vitro recombination method

The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonuclease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

In vitro recombination method

Young, Lei↗

DOE OSTI2020

Methods of introducing nucleic acids into cellular DNA

A method of introducing a nucleic acid sequence into a cell is provided where the cell has impaired or inhibited or disrupted DnaG primase activity or impaired or inhibited or disrupted DnaB helicase activity, or larger or increased gaps or distance between Okazaki fragments or lowered or reduced frequency of Okazaki fragment initiation, or the cell has increased single stranded DNA (ssDNA) on the lagging strand of the replication fork including transforming the cell through recombination with a nucleic acid oligomer.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

The crystal structure of bacteriophage λ RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins

Abstract RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the λ bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here, we present the 2.05-Å crystal structure of the λ RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron–electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.

Biochemistry & Molecular Biology↗

DOE OSTI2025

RTx-303, an Orally Bioavailable Polθ Polymerase Inhibitor That Potentiates PARP Inhibitors in BRCA Mutant Tumors

Abstract DNA polymerase θ (Polθ) is a polymerase-helicase fusion protein that is synthetically lethal with homologous recombination (HR) factors, such as BRCA1/2, and confers resistance to PARP inhibitors (PARPi) and other genotoxic cancer therapies. Previously developed Polθ polymerase (Polθ-pol) inhibitors (Polθi) exhibited limited pharmacological activity and metabolic stability, warranting the development of a Polθi with improved drug-like properties. Here, we developed RTx-303, a selective allosteric small-molecule Polθ-pol inhibitor that exhibits 5.1 nM IC50, 88% oral bioavailability, and a prolonged half-life along with its equipotent metabolite. X-ray crystallography highlights the development of a solvent-exposed side-chain that is essential for the optimal drug-like properties of RTx-303. Notably, RTx-303 exhibits significantly higher cellular potency than previously developed Polθ-pol inhibitors and strongly potentiates PARPi in BRCA1/2 mutant cells and patient-derived xenograft models. The superior potency, robust pharmacological activity, and high tolerability of RTx-303 warrant further development as a Polθ-pol inhibitor drug candidate.

Chandramouly, Gurushankar [Thomas Jefferson Univer↗

DOE OSTI2022

High-resolution mapping reveals hotspots and sex-biased recombination in Populus trichocarpa

Abstract Fine-scale meiotic recombination is fundamental to the outcome of natural and artificial selection. Here, dense genetic mapping and haplotype reconstruction were used to estimate recombination for a full factorial Populus trichocarpa cross of 7 males and 7 females. Genomes of the resulting 49 full-sib families (N = 829 offspring) were resequenced, and high-fidelity biallelic SNP/INDELs and pedigree information were used to ascertain allelic phase and impute progeny genotypes to recover gametic haplotypes. The 14 parental genetic maps contained 1,820 SNP/INDELs on average that covered 376.7 Mb of physical length across 19 chromosomes. Comparison of parental and progeny haplotypes allowed fine-scale demarcation of cross-over regions, where 38,846 cross-over events in 1,658 gametes were observed. Cross-over events were positively associated with gene density and negatively associated with GC content and long-terminal repeats. One of the most striking findings was higher rates of cross-overs in males in 8 out of 19 chromosomes. Regions with elevated male cross-over rates had lower gene density and GC content than windows showing no sex bias. High-resolution analysis identified 67 candidate cross-over hotspots spread throughout the genome. DNA sequence motifs enriched in these regions showed striking similarity to those of maize, Arabidopsis, and wheat. These findings, and recombination estimates, will be useful for ongoing efforts to accelerate domestication of this and other biomass feedstocks, as well as future studies investigating broader questions related to evolutionary history, perennial development, phenology, wood formation, vegetative propagation, and dioecy that cannot be studied using annual plant model systems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Strategies to identify and edit improvements in synthetic genome segments episomally

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments at low copy number. However, all stages of whole-genome engineering have the potential to impose mutations on the synthetic genome that can reduce or eliminate the fitness of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to recoding an Escherichia coli strain to replace seven codons with synonymous alternatives genome wide. Ten 44 402–47 179 bp de novo synthesized DNA segments contained in a BAC from the recoded strain were unable to complement deletion of the corresponding 33–61 wild-type genes using a single antibiotic resistance marker. Next-generation sequencing (NGS) was used to identify 1–7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the recoded segment contained in the BAC when both the recoded and wild-type copies of the mutated genes had to exist by necessity during the repair process. Finally, two web-based tools were used to predict the impact of a subset of non-recoding missense mutations on strain fitness using protein structure and function calls.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Differential roles of kinetic on- and off-rates in T-cell receptor signal integration revealed with a modified Fab’-DNA ligand

Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide major histocompatibility complex (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRβ H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab’-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rates of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells, which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from TCR ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient.

Science & Technology - Other Topics↗

DOE OSTI2022

Novel cis-regulatory elements as synthetic promoters to drive recombinant protein expression from the Chlamydomonas reinhardtii nuclear genome

Eukaryotic green microalgae represent a sustainable, photosynthetic biotechnology platform for generating high-value products. The model green alga Chlamydomonas reinhardtii has already been used to generate high value bioproducts such as recombinant proteins and terpenoids. However, low, unstable, and variable nuclear transgene expression has limited the ease and speed of metabolic engineering and recombinant protein expression in this system. Here, novel genetic devices for transgene expression in C. reinhardtii have been developed by identifying cis-regulatory DNA elements capable of driving high transgene expression in C. reinhardtii promoters using de novo motif discovery informatics approaches. Thirteen putative motifs were synthesized as concatemers, linked to a common minimal basal promoter, and assayed for their activity to drive expression of a yellow fluorescent protein reporter gene. Following transformation of the vectors into C. reinhardtii by electroporation, in vivo measurements of yellow fluorescent protein expression by flow cytometry revealed that five of the DNA motifs analyzed displayed significantly higher reporter expression compared to the basal promoter control. Two of the concatemerized motifs, despite being much smaller minimal cis-regulatory elements, drove reporter expression at levels approaching that of the conventionally-used AR1 promoter. This analysis provides insight into C. reinhardtii promoter structure and gene regulation, and provides a new toolbox of cis-regulatory elements that can be used to drive transgene expression at a variety of expression levels.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Overexpression of human ACE2 protein in mouse fibroblasts stably transfected with the intact ACE2 gene

Infection by SARS-CoV-2 is dependent on binding of the viral spike protein to angiotensin converting enzyme 2 (ACE2), a membrane glycoprotein expressed on epithelial cells in the human upper respiratory tract. Recombinant ACE2 protein has potential application for anti-viral therapy. Here we co-transfected mouse fibroblasts (A9 cells) with a cloned fragment of human genomic DNA containing the intact ACE2 gene and an unlinked neomycin phosphotransferase gene, and then selected stable neomycin-resistant transfectants. Transfectant clones expressed ACE2 protein at levels that were generally proportional to the number of ACE2 gene copies integrated in the cell genome, ranging up to approximately 50 times the level of ACE2 present of Vero-E6 cells. Cells overexpressing ACE2 were hypersensitive to infection by spike-pseudotyped vesicular stomatitis virus (VSV-S), and adsorption of VSV-S to these cells occurred at an accelerated rate compared to Vero-E6 cells. In conclusion, the transfectant cell clones described here therefore have favorable attributes as feedstocks for large-scale production of recombinant human ACE2 protein.

77 NANOSCIENCE AND NANOTECHNOLOGY↗