DNA Rearrangements Research Articles

Distinct Classes of Complex Structural Variation Uncovered across Thousands of Cancer Genome Graphs

Cancer genomes often harbor hundreds of somatic DNA rearrangement junctions, many of which cannot be easily classified into simple (e.g., deletion) or complex (e.g., chromothripsis) structural variant classes. Applying a novel genome graph computational paradigm to analyze the topology of junction copy number (JCN) across 2,778 tumor whole-genome sequences, we uncovered three novel complex rearrangement phenomena: pyrgo, rigma, and tyfonas. Pyrgo are "towers" of low-JCN duplications associated with early-replicating regions, superenhancers, and breast or ovarian cancers. Rigma comprise "chasms" of low-JCN deletions enriched in late-replicating fragile sites and gastrointestinal carcinomas. Tyfonas are "typhoons" of high-JCN junctions and fold-back inversions associated with expressed protein-coding fusions, breakend hypermutation, and acral, but not cutaneous, melanomas. Clustering of tumors according to genome graph-derived features identified subgroups associated with DNA repair defects and poor prognosis.

NRG1 fusion-driven tumors: biology, detection, and the therapeutic role of afatinib and other ErbB-targeting agents.

Oncogenic gene fusions are hybrid genes that result from structural DNA rearrangements, leading to deregulated activity. Fusions involving the neuregulin-1 gene (NRG1) result in ErbB-mediated pathway activation and therefore present a rational candidate for targeted treatment. The most frequently reported NRG1 fusion is CD74-NRG1, which most commonly occurs in patients with invasive mucinous adenocarcinomas (IMAs) of the lung, although several other NRG1 fusion partners have been identified in patients with lung cancer, including ATP1B1, SDC4, and RBPMS. NRG1 fusions are also present in patients with other solid tumors, such as pancreatic ductal adenocarcinoma. In general, NRG1 fusions are rare across different types of cancer, with a reported incidence of <1%, with the notable exception of IMA, which represents ≈2%-10% of lung adenocarcinomas and has a reported incidence of ≈10%-30% for NRG1 fusions. A substantial proportion (≈20%) of NRG1 fusion-positive non-small-cell lung cancer cases are nonmucinous adenocarcinomas. ErbB-targeted treatments, such as afatinib, a pan-ErbB tyrosine kinase inhibitor, are potential therapeutic strategies to address unmet treatment needs in patients harboring NRG1 fusions.

Botanical and molecular alterations in some strawberry cultivars caused by some soil-borne fungi

Charcoal rot caused are the most dangerous soil-borne diseases that infect strawberry in Egypt. These pathogens have mycotoxic activities; this encouraged us to study the effect of these activities on some botanical traits and at molecular level using the start codon targeted (SCoT) markers technique. Where infection with pathogens led to highly decrease in yield, all vegetative growth characteristics were affected in all cultivars. The infection had a positive effect on the disease incidence and severity in all cultivars. Five SCoT primers proved to be valid in showing molecular alterations between infected and uninfected plants, which may be due to the structural rearrangements in DNA by mutagenic activity for mycotoxins. Cluster analysis showed molecular and phenotypic variation among infected plants and uninfected plants. Fungal infections induced molecular alterations with an average of 41.3% polymorphism. The results give an indication of the presence of mutagenic effects for Charcoal rot on strawberry cultivars under study.

Comparative Analysis of Streptococcus pneumoniae Type I Restriction-Modification Loci: Variation in hsdS Gene Target Recognition Domains.

Streptococcus pneumoniae (pneumococcus) is a respiratory commensal pathogen that causes a range of infections, particularly in young children and the elderly. Pneumococci undergo spontaneous phase variation in colony opacity phenotype, in which DNA rearrangements within the Type I restriction-modification (R-M) system specificity gene hsdS can potentially generate up to six different hsdS alleles with differential DNA methylation activity, resulting in changes in gene expression. To gain a broader perspective of this system, we performed bioinformatic analyses of Type I R-M loci from 18 published pneumococcal genomes, and one R-M locus sequenced for this study, to compare genetic content, organization, and homology. All 19 loci encoded the genes hsdR, hsdM, hsdS, and at least one hsdS pseudogene, but differed in gene order, gene orientation, and hsdS target recognition domain (TRD) content. We determined the coding sequences of 87 hsdS TRDs and excluded seven from further analysis due to the presence of premature stop codons. Comparative analyses revealed that the TRD 1.1, 1.2, and 2.1 protein sequences had single amino acid substitutions, and TRD 2.2 and 2.3 each had seven differences. The results of this study indicate that variability exists among the gene content and arrangements within Type I R-M loci may provide an additional level of divergence between pneumococcal strains, such that phase variation-mediated control of virulence factors may vary significantly between individual strains. These findings are consistent with presently available transcript profile data.

Altered 3D chromatin structure permits inversional recombination at the IgH locus.

Immunoglobulin heavy chain (IgH) genes are assembled by two sequential DNA rearrangement events that are initiated by recombination activating gene products (RAG) 1 and 2. Diversity (DH) gene segments rearrange first, followed by variable (VH) gene rearrangements. Here, we provide evidence that each rearrangement step is guided by different rules of engagement between rearranging gene segments. DH gene segments, which recombine by deletion of intervening DNA, must be located within a RAG1/2 scanning domain for efficient recombination. In the absence of intergenic control region 1, a regulatory sequence that delineates the RAG scanning domain on wild-type IgH alleles, VH and DH gene segments can recombine with each other by both deletion and inversion of intervening DNA. We propose that VH gene segments find their targets by distinct mechanisms from those that apply to DH gene segments. These distinctions may underlie differential allelic choice associated with each step of IgH gene assembly.

Abstract 184: SureSelect sequencing panels and algorithms to detect copy number variations (CNVs), DNA rearrangements, microsatellite Instability and tumor mutational burden (TMB) in FFPE specimens

Abstract Background: The current standard of genomic profiling of cancer tissues relies upon multiple separate technologies to aid in tumor characterization. Here we describe an NGS panel and analysis workflow for cancer samples that can detect single nucleotide polymorphisms (SNPs), insertions and deletions (INDEL), somatic copy number alterations (SCNAs), and translocations (TLs), Tumor Mutation Burden (TMB) and Microsatellite Instability (MSI) in a single assay. Methods: We have developed a SureSelect target enrichment sequencing panels for comprehensive profiling of single nucleotide variations (SNP/INDEL), gene copy number variations (CNV), and DNA translocations (TL) and relevant microsatellite regions in a single assay. The TMB panel gene list was curated based on genes found in cancer gene databases such as Clinical Interpretations of Variants in Cancer (CIViC), Cancer Genome Interpreter (CGIdb), Catalog of Somatic Mutations in Cancer Census (COSMIC), Precision Oncology Knowledge Base (OncoKB). An accompanying data analysis pipeline will provide highly sensitive and accurate detection of variants and allows for the determination TMB and MSI status. Probes have been selected for over 500 genes to detect SCNAs and translocation in several cancer subtypes. The probes are optimized with the SureSelect XTHS protocol, which enables sensitive and accurate detection of rare mutation events within a heterogeneous sample with molecular barcode-mediated error correction. The SCNAs probes target regions in the genome that help in decreasing the noise for SCNAs calling. The data analysis utilizes either a matched normal or standard control DNA to compute read depth log ratios. Aberrant regions of constant ploidy are first identified followed by the application of a variational streaming algorithm on log ratio and SNP allele frequencies to determine the major clones present in the tumor. Translocation detection was verified on lung cancer samples by analyzing split reads across the fusion breakpoints. Results: By diluting samples with known copy-number aberrations into normal DNA, a limit of detection of ⇐2.3 copies/cell was demonstrated, equivalent to the detection of 3 copies at 30% tumor fraction. A similar approach demonstrated an ability to detect EML4-ALK and SLC24A2-ROS1 translocations at 3% allele frequency. Performance of the assay was further evaluated using 100+ FFPE samples harboring rearrangements, gene amplifications, and SNP/Indels. Concordance to the reference methods (FISH, WES, WGS) was &amp;gt;90%. TMB and MSI algorithms were benchmarked using in silico analysis of TCGA data and performance of the assay was evaluated on FFPE samples with orthogonally-determined MSI/TMB status. Conclusions: This work represents an important advancement in the development of a single assay to detect copy number variation, DNA rearrangement and mutations, TMB and MSI status of a FFPE sample. Citation Format: Arjun Vadapalli, Akanksha Khare, Hanjun Shin, Ashutosh Ashutosh, Linus Forsmark, Anne Lucas, Scott Happe, Gilbert Amparo, Carlos Pabon, Jayati Ghosh, Tracy Liu, Jimmy Jin, Mike Ruvolo, Douglas Roberts. SureSelect sequencing panels and algorithms to detect copy number variations (CNVs), DNA rearrangements, microsatellite Instability and tumor mutational burden (TMB) in FFPE specimens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 184.

SIGAR: Inferring Features of Genome Architecture and DNA Rearrangements by Split-Read Mapping.

Ciliates are microbial eukaryotes with distinct somatic and germline genomes. Postzygotic development involves extensive remodeling of the germline genome to form somatic chromosomes. Ciliates therefore offer a valuable model for studying the architecture and evolution of programed genome rearrangements. Current studies usually focus on a few model species, where rearrangement features are annotated by aligning reference germline and somatic genomes. Although many high-quality somatic genomes have been assembled, a high-quality germline genome assembly is difficult to obtain due to its smaller DNA content and abundance of repetitive sequences. To overcome these hurdles, we propose a new pipeline, SIGAR (Split-read Inference of Genome Architecture and Rearrangements) to infer germline genome architecture and rearrangement features without a germline genome assembly, requiring only short DNA sequencing reads. As a proof of principle, 93% of rearrangement junctions identified by SIGAR in the ciliate Oxytricha trifallax were validated by the existing germline assembly. We then applied SIGAR to six diverse ciliate species without germline genome assemblies, including Ichthyophthirius multifilii, a fish pathogen. Despite the high level of somatic DNA contamination in each sample, SIGAR successfully inferred rearrangement junctions, short eliminated sequences, and potential scrambled genes in each species. This pipeline enables pilot surveys or exploration of DNA rearrangements in species with limited DNA material access, thereby providing new insights into the evolution of chromosome rearrangements.

Selfing mutants link Ku proteins to mating type determination in Tetrahymena.

Recognition of self and nonself is important for outcrossing organisms, and different mating types establish the barrier against self-mating. In the unicellular ciliate T. thermophila, mating type determination requires complex DNA rearrangements at a single mat locus during conjugation to produce a type-specific gene pair (MTA and MTB) for 1 of 7 possible mating types. Surprisingly, we found that decreased expression of the DNA breakage-repair protein Ku80 at late stages of conjugation generated persistent selfing phenotype in the progeny. DNA analysis revealed multiple mating-type gene pairs as well as a variety of mis-paired, unusually arranged mating-type genes in these selfers that resemble some proposed rearrangement intermediates. They are found also in normal cells during conjugation and are lost after 10 fissions but are retained in Ku mutants. Silencing of TKU80 or TKU70-2 immediately after conjugation also generated selfing phenotype, revealing a hidden DNA rearrangement process beyond conjugation. Mating reactions between the mutant and normal cells suggest a 2-component system for self–nonself-recognition through MTA and MTB genes.

Site-Specific Recombination with Inverted Target Sites: A Cautionary Tale of Dicentric and Acentric Chromosomes.

Site-specific recombinases are widely used tools for analysis of genetics, development, and cell biology, and many schemes have been devised to alter gene expression by recombinase-mediated DNA rearrangements. Because the FRT and lox target sites for the commonly used FLP and Cre recombinases are asymmetrical, and must pair in the same direction to recombine, construct design must take into account orientation of the target sites. Both direct and inverted configurations have been used. However, the outcome of recombination between target sites on sister chromatids is frequently overlooked. This is especially consequential with inverted target sites, where exchange between oppositely oriented target sites on sisters will produce dicentric and acentric chromosomes. By using constructs that have inverted target sites in Drosophila melanogaster and in mice, we show here that dicentric chromosomes are produced in the presence of recombinase, and that the frequency of this event is quite high. The negative effects on cell viability and behavior can be significant, and should be considered when using such constructs.

Identification of genetic variants controlling RNA editing and their effect on RNA structure stabilization.

Post-transcriptional modification of RNA (RNA editing, RNAe) results in differences between the RNA transcript and the genomic DNA sequence (RDD). Enzymatic modification of adenosine to inosine (A2I) by ADAR is the most studied type of RNAe. However, few genetic association studies with A2I RNAe events have been conducted. Some studies have analyzed the inter-population RNAe-QTL diversity in humans, but the sample size of these studies was limited. Other types of RNA and DNA differences have been reported but are largely understudied. Here, we report a comprehensive analysis of all types of RDD, based on two independent datasets. We found that A2I was by far the most observed type of RDD. Moreover, manual curation suggests that A2I is likely the only enzymatically driven RNAe type observed in blood derived DNA, all other non-A2I RDD could either be attributed to sequencing and processing artifacts, or are a result of somatic DNA rearrangements. We then conducted an in-cis genetic association study and identified 472 genetic associations (RNAe-QTL), that were replicated in both datasets. We confirm the potential effect of the RNAe-QTL on RNA structure by showing that allele specific RNAe occurs in heterozygotes. Although the generally assumed function of RNAe is to destabilize double stranded RNA structure, we found clear evidence for the potential additional involvement of RNAe in maintaining RNA hairpin that has been altered by the RNAe-QTL. Our study confirms, in two independent datasets, the potential role of RNAe in maintaining RNA structure in the presence of genetic variation.

Clinical Phenotype and Genetic Features of a Pair of Chinese Twins with Kearns-Sayre Syndrome.

Kearns-Sayre Syndrome (KSS) is a severe mitochondrial disorder involving the central nervous system, eyes, ears, skeletal muscles, and heart. The mitochondrial DNA (mtDNA) rearrangements, especially the deletions, are present in almost all KSS patients and considered as the disease-causing factor. However, the size and position of mtDNA deletions are distinct in different individuals. In this study, we report the case of a pair of Chinese twins with KSS. The twin patients revealed typical KSS clinical symptoms, including heart block, bilateral sensorineural hearing loss, progressive external ophthalmoplegia, exercise intolerance, proximal limb weakness, and endocrine disorders. Using long-range polymerase chain reactions (long-range PCR) and next-generation sequencing (NGS), the genetic features of the twin patients were investigated. A large 6600 bp mtDNA deletion, ranging from position 8702 to 15,302, was detected in both patients. To our knowledge, this kind of mtDNA deletion has never been described previously. Our study enriched the mutation spectrum of KSS and showed that NGS is a powerful tool for detecting mtDNA large variants.

Clinical and cytopathological characteristics of HTLV-1+ hodgkin lymphoma.

BackgroundHuman T‐lymphotropic virus‐1 (HTLV‐1)+ Hodgkin lymphoma (HL) is difficult to differentiate from adult T‐cell leukemia/lymphoma (ATLL) with HL‐like histology (HL‐like ATLL).MethodsCytological and immunohistological features, HTLV‐1 proviral DNA integration, and rearrangements of the T‐cell receptor (TCR) Cβ1 gene were examined in 11 HTLV‐1+ patients with HL‐like disease.ResultsSix patients were classified as HTLV‐1+ HL and five as HL‐like ATLL in accordance with genetic findings of HTLV‐1 proviral DNA integration and rearrangements of the TCR Cβ1 gene. Small ordinary looking lymphocytes with round nuclei were detected in the background of six patients with HTLV‐1+ HL, which were immunohistochemically negative for CD25 and CC chemokine receptor (CCR)4 and had a low MIB1 labeling index (mean: 28.3%). In the HL‐like ATLL specimens, small‐ and medium‐sized atypical lymphocytes with indented and irregular‐shaped nuclei were found, and were diffusely positive for CD25 and CCR4, with high MIB1 labeling (mean: 76%). Both groups had scattered CD30+ and CD15+ Hodgkin and Reed Sternberg (RS) giant cells, with or without CD20 expression and Epstein‐Barr virus infection. The 50% overall survival period was significantly longer for the HTLV‐1+ HL group (180 months) than for the HL‐like ATLL group (7.8 months; P = .004).ConclusionsHTLV‐1+ HL showed typical small lymphoid cells with a low MIB1 labeling index in a background of Hodgkin and RS cells, with some scattered CD25+ and CCR4+ lymphocytes. In HTLV‐1 endemic areas, distinguishing HTLV‐1+ HL from HL‐like ATLL is important because of their differing treatment strategies and prognoses.

Low-temperature pretreatment of explants and high maltose concentration during callus culture improves particle-bombardment-mediated stable transgene expression in common wheat.

Biolistic transformation systems are widely used to introduce foreign genes into common wheat (Triticum aestivum L.); however, these techniques often generate high transgene copy numbers and complex transgene integration patterns that hinder the stable expression of the transgenes. To improve the efficiency of stable transgene expression, we examined the effect of low-temperature pretreatment of wheat flower spikes and of high maltose concentration (HMC) in the medium during the subsequent callus culture. Tillers of the spring wheat cultivar Bobwhite were stored at 5°C without water for one week before the isolation of their immature scutellar tissues, and the resulting particle-bombarded explants were cultured on 15% maltose for a month. Together, these treatments significantly increased the number of recovered transgenic lines expressing the reporter gene. The low-temperature pretreatment eliminated the negative effects of HMC, and HMC improved the efficiency of stable transgene expression. Southern blot analysis revealed that transgenic lines recovered after HMC treatment integrated a lower copy number of transgenes than those cultured at normal (4%) maltose concentration. These findings suggest that the HMC-mediated reduction of the transgene copy number results from the suppression of plasmid DNA rearrangement before or during transgene integration into the wheat genome.

Abstract A15: Detection of genome-wide copy number alterations in tumor tissue and cell-free DNA of pancreatic cancer patients

Abstract Pancreatic cancer is considered one of the deadliest malignancies, with only an 8% 5-year survival. The diagnosis of pancreatic cancer is very challenging since early-stage pancreatic cancer is associated with nonspecific and rarely noticeable symptoms. Additionally, the current diagnostic tools (such as imaging and the measurement of serum tumor marker CA19-9 levels) have limitations. Liquid biopsies make it possible to detect tumor-specific molecular alterations by analysis of cell-free DNA (cfDNA) isolated from plasma, which contains circulating tumor DNA (ctDNA). Large genomic rearrangements have become evident as key mutagenic events in the progression of solid tumors. In this study we evaluated the prevalence of large-scale genomic rearrangements in pancreatic resections and cell-free DNA (cfDNA) of pancreatic cancer patients. Genome-wide copy number profiles were derived from shallow whole-genome sequencing (sWGS). We applied sWGS to cfDNA samples from all patients at different time points. Concordance of the profiles was evaluated between cfDNA and matched FFPE tumor tissue in samples of operable pancreatic cancer patients. Clear copy number variations (CNVs) were observed in 13 FFPE tumor samples of 16 resectable patients (81%). This indicates that chromosomal instability is a frequent mechanism in pancreatic cancer. We then evaluated if CNVs could be detected in cfDNA of operable patients. Unfortunately, in none of the 50 operable patients were CNVs established. In contrast, cfDNA analysis in metastatic patients revealed clear CNVs in 66.7% (10/15) of the patients at the time of diagnosis. Although cfDNA concentrations were not statistically significantly increased compared to operable patients, remarkably higher CA19-9 values were determined in nonoperable patients. Preliminary data indicate that this approach can predict recurrence. For instance, a copy number change in a follow-up sample (1-year post-surgery; 6-months post-chemotherapy) was observed in a borderline-operative patient diagnosed with a liver metastasis. The CNV profiles obtained in both FFPE resections as well as in cfDNA were patient unique, but some recurrent alterations on chromosome 9 (region with CDKN2A and CDKN2B) and on chromosome 12 (region with KRAS) were ascertained. These results demonstrate detection of tumor-derived CNVs in cfDNA of advanced pancreatic cancer cases. We also show that there is potential to use cfDNA-derived profiles to monitor treatment response. Based on the results of this proof-of-concept study, additional patients are currently being analyzed. Citation Format: Greet Wieme, Frederik Berrevoet, Aude Vanlander, Jo Van Dorpe, Anne Hoorens, Bram Parton, Jurgen Van Limmen, Ann De Bruyne, Marc De Man, Karen Geboes, Kathleen Claes. Detection of genome-wide copy number alterations in tumor tissue and cell-free DNA of pancreatic cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A15.

Discovery of Four Novel ORFs Responsible for Cytoplasmic Male Sterility (CMS) in Cotton (Gossypium hirsutum L.) through Comparative Analysis of the Mitochondrial Genomes of Four Isoplasmic Lines

Cytoplasmic male sterility (CMS) is an important feature for achieving heterosis in the development of hybrid crops. Mitochondria contribute to CMS, especially via mitochondrial DNA (mtDNA) rearrangements and chimeric genes. However, the mechanisms of CMS have not been fully elucidated, and the isonuclear alloplasmic lines used in previous studies have limited utility in cotton CMS research. In this study, three CMS lines (J4A-1, J4A-2 and J4A-3) and their isoplasmic maintainer line (J4B) were analyzed for mtDNA structural differences via high-throughput sequencing. The results showed that mtDNA was conserved (with similarities higher than 99%) among the three CMS lines and their isoplasmic maintainer line. All lines harbored 36 known protein-coding genes, 3 rRNAs, and 15 tRNAs. The protein-coding genes with non-synonymous mutations mainly encoded two types of proteins: ATPase and ribosomal proteins. Four new open reading frames (ORFs) (orf116b, orf186a-1, orf186a-2 and orf305a) were identified as candidate ORFs responsible for CMS. Two of the ORFs (orf186a-1 and orf186a-2) were identified as orf4 and orf4-2 of the upland cotton CMS line 2074A (a line with Gossypium harknessii Brandegee CMS-D2-2 cytoplasm), respectively. These findings provide a reference for CMS research in cotton or other crops.

Three-dimensional genome rewiring during the development of antibody-secreting cells.

The development of B lymphocytes into antibody-secreting plasma cells is central to the adaptive immune system in that it confers protective and specific antibody response against invading pathogen. This developmental process involves extensive morphological and functional alterations that begin early after antigenic stimulation. These include chromatin restructuring that is critical in regulating gene expression, DNA rearrangement and other cellular processes. Here we outline the recent understanding of the three-dimensional architecture of the genome, specifically focused on its contribution to the process of B cell activation and terminal differentiation into antibody-secreting cells.

Unique Features of Immunity within the Immunoglobulin Heavy Chain Locus of Egyptian Rousette Bats

Abstract The Egyptian fruit bat (Rousettus aegyptiacus, ERB) is reported to be the reservoir of Marburg virus (MARV). Indeed, MARV infection is almost completely asymptomatic in ERBs while eliciting a severe hemorrhagic fever in humans and non-humans primates (NHPs). To understand the biological mechanisms that explain these differential outcomes to MARV, we aim to investigate the differences between both animals’ immune systems. One of the antiviral mechanisms to control viruses is the humoral response. The B cell receptor (BCR) is composed of two heavy and two light chains heterodimers. Both immunoglobulin (Ig) chains undergo DNA rearrangement to generate a diverse repertoire, crucial for diverse antigenic recognition. The Ig heavy chain locus is comprised of the variable (V), diversity (D), joining (J), and constant (C) genes. Utilizing high throughput sequencing to resolve the V(D)J rearrangements we can characterize the BCR repertoire and define the clonotypes that are generated in response to vaccination or infection. To define the BCR repertoire with DNA sequencing, a thorough representation of the Ig heavy chain (IGH) locus is necessary. We were able to resolve on contiguous IGH locus containing all BCR genes that were previously on 21 different scaffolds. In addition, we identified a unexpected expansion of IgE genes not described in another species to date. We defined the tissue expression of these novel IgE genes as well as IgGs, IgA, and IgM. Finally, we predicted differential functions between subclasses in silico. These data support unique immune response capabilities of ERBs that may aid in their control of pathogenic human viruses.

Altered 3D chromatin structure permits inversional recombination at the IgH locus

Abstract Immunoglobulin heavy chain (IgH) genes are assembled by two sequential DNA rearrangement events that are initiated by recombinase activating gene products (RAG) 1 and 2. Diversity gene segments (DH) rearrange first, followed by variable (VH) gene rearrangements. Here we provide evidence that each rearrangement step is guided by different rules of engagement between rearranging gene segments. DH gene segments, that recombine by deletion of intervening DNA, must be located within a RAG1/2 scanning domain for efficient recombination. In the absence of intergenic control region 1, a regulatory sequence that delineates the RAG scanning domain on WT IgH alleles, VH and DH gene segments can recombine with each other by both deletion and inversion of intervening DNA. We propose that VH gene segments find their targets by diffusion-controlled mechanisms. These distinct mechanisms may underlie differential allelic choice associated with each step of IgH gene assembly.

Chiral Carbon Dots Mimicking Topoisomerase I To Mediate the Topological Rearrangement of Supercoiled DNA Enantioselectively.

Nanomaterials with enzyme-mimetic activities are possible alternatives to natural enzymes. Mimicking enzymatic enantioselectivity remains a great challenge. Herein, we report that cysteine-derived chiral carbon dots (CDs) can mimic topoisomerase I to mediate topological rearrangement of supercoiled DNA enantioselectively. d-CDs can more effectively catalyze the topological transition of plasmid DNA from supercoiled to nicked open-circular configuration than l-CDs. Experiments suggest the underlying mechanism: d-CDs intercalatively bind with DNA double helix more strongly than l-CDs; the intercalative CDs can catalyze the production of hydroxyl radicals to cleave phosphate backbone in one strand of the double helix, leading to topological rearrangement of supercoiled DNA. Molecular dynamics (MD) simulation show that the stronger affinity for hydrogen-bond formation and hydrophobic interaction between d-cysteine and DNA than that of l-cysteine is the origin of enantioselectivity.

Chiral Carbon Dots Mimicking Topoisomerase I To Mediate the Topological Rearrangement of Supercoiled DNA Enantioselectively

AbstractNanomaterials with enzyme‐mimetic activities are possible alternatives to natural enzymes. Mimicking enzymatic enantioselectivity remains a great challenge. Herein, we report that cysteine‐derived chiral carbon dots (CDs) can mimic topoisomerase I to mediate topological rearrangement of supercoiled DNA enantioselectively. d‐CDs can more effectively catalyze the topological transition of plasmid DNA from supercoiled to nicked open‐circular configuration than l‐CDs. Experiments suggest the underlying mechanism: d‐CDs intercalatively bind with DNA double helix more strongly than l‐CDs; the intercalative CDs can catalyze the production of hydroxyl radicals to cleave phosphate backbone in one strand of the double helix, leading to topological rearrangement of supercoiled DNA. Molecular dynamics (MD) simulation show that the stronger affinity for hydrogen‐bond formation and hydrophobic interaction between d‐cysteine and DNA than that of l‐cysteine is the origin of enantioselectivity.