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Genome Research Jul 2023The assay for transposase-accessible chromatin with sequencing (ATAC-seq) is a common assay to identify chromatin accessible regions by using a Tn5 transposase that can...
The assay for transposase-accessible chromatin with sequencing (ATAC-seq) is a common assay to identify chromatin accessible regions by using a Tn5 transposase that can access, cut, and ligate adapters to DNA fragments for subsequent amplification and sequencing. These sequenced regions are quantified and tested for enrichment in a process referred to as "peak calling." Most unsupervised peak calling methods are based on simple statistical models and suffer from elevated false positive rates. Newly developed supervised deep learning methods can be successful, but they rely on high quality labeled data for training, which can be difficult to obtain. Moreover, though biological replicates are recognized to be important, there are no established approaches for using replicates in the deep learning tools, and the approaches available for traditional methods either cannot be applied to ATAC-seq, where control samples may be unavailable, or are post hoc and do not capitalize on potentially complex, but reproducible signal in the read enrichment data. Here, we propose a novel peak caller that uses unsupervised contrastive learning to extract shared signals from multiple replicates. Raw coverage data are encoded to obtain low-dimensional embeddings and optimized to minimize a contrastive loss over biological replicates. These embeddings are passed to another contrastive loss for learning and predicting peaks and decoded to denoised data under an autoencoder loss. We compared our replicative contrastive learner (RCL) method with other existing methods on ATAC-seq data, using annotations from ChromHMM genomic labels and transcription factor ChIP-seq as noisy truth. RCL consistently achieved the best performance.
Topics: Chromatin Immunoprecipitation Sequencing; Sequence Analysis, DNA; High-Throughput Nucleotide Sequencing; Chromatin; DNA
PubMed: 37217250
DOI: 10.1101/gr.277677.123 -
Journal of Molecular Biology Dec 2020More than a million Okazaki fragments are synthesized, processed and joined during replication of the human genome. After synthesis of an RNA-DNA oligonucleotide by DNA...
More than a million Okazaki fragments are synthesized, processed and joined during replication of the human genome. After synthesis of an RNA-DNA oligonucleotide by DNA polymerase α holoenzyme, proliferating cell nuclear antigen (PCNA), a homotrimeric DNA sliding clamp and polymerase processivity factor, is loaded onto the primer-template junction by replication factor C (RFC). Although PCNA interacts with the enzymes DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (LigI) that complete Okazaki fragment processing and joining, it is not known how the activities of these enzymes are coordinated. Here we describe a novel interaction between Pol δ and LigI that is critical for Okazaki fragment joining in vitro. Both LigI and FEN1 associate with PCNA-Pol δ during gap-filling synthesis, suggesting that gap-filling synthesis is carried out by a complex of PCNA, Pol δ, FEN1 and LigI. Following ligation, PCNA and LigI remain on the DNA, indicating that Pol δ and FEN1 dissociate during 5' end processing and that LigI engages PCNA at the DNA nick generated by FEN1 and Pol δ. Thus, dynamic PCNA complexes coordinate Okazaki fragment synthesis and processing with PCNA and LigI forming a terminal structure of two linked protein rings encircling the ligated DNA.
Topics: DNA; DNA Ligase ATP; DNA Ligases; DNA Polymerase I; DNA Polymerase III; DNA Replication; Flap Endonucleases; Genome, Human; Holoenzymes; Humans; Multiprotein Complexes; Proliferating Cell Nuclear Antigen; Protein Binding; Replication Protein C
PubMed: 33157085
DOI: 10.1016/j.jmb.2020.10.032 -
Genes Oct 2019DNA topoisomerase II (TOP2) plays a critical role in many processes such as replication and transcription, where it resolves DNA structures and relieves torsional... (Review)
Review
DNA topoisomerase II (TOP2) plays a critical role in many processes such as replication and transcription, where it resolves DNA structures and relieves torsional stress. Recent evidence demonstrated the association of TOP2 with topologically associated domains (TAD) boundaries and CCCTC-binding factor (CTCF) binding sites. At these sites, TOP2 promotes interactions between enhancers and gene promoters, and relieves torsional stress that accumulates at these physical barriers. Interestingly, in executing its enzymatic function, TOP2 contributes to DNA fragility through re-ligation failure, which results in persistent DNA breaks when unrepaired or illegitimately repaired. Here, we discuss the biological processes for which TOP2 is required and the steps at which it can introduce DNA breaks. We describe the repair processes that follow removal of TOP2 adducts and the resultant broken DNA ends, and present how these processes can contribute to disease-associated mutations. Furthermore, we examine the involvement of TOP2-induced breaks in the formation of oncogenic translocations of leukemia and papillary thyroid cancer, as well as the role of TOP2 and proteins which repair TOP2 adducts in other diseases. The participation of TOP2 in generating persistent DNA breaks and leading to diseases such as cancer, could have an impact on disease treatment and prevention.
Topics: CCCTC-Binding Factor; Chromatin; DNA; DNA Breaks, Double-Stranded; DNA Repair; DNA Topoisomerases, Type II; Humans; Leukemia, Myeloid, Acute; Thyroid Cancer, Papillary; Topoisomerase II Inhibitors; Torsion, Mechanical
PubMed: 31614754
DOI: 10.3390/genes10100791 -
Biochemical Society Transactions Dec 2019Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), which is the most toxic DNA damage in cells. Unrepaired DSBs can cause... (Review)
Review
Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), which is the most toxic DNA damage in cells. Unrepaired DSBs can cause genome instability, tumorigenesis or cell death. DNA end synapsis is the first and probably the most important step of the NHEJ pathway, aiming to bring two broken DNA ends close together and provide structural stability for end processing and ligation. This process is mediated through a group of NHEJ proteins forming higher-order complexes, to recognise and bridge two DNA ends. Spatial and temporal understanding of the structural mechanism of DNA-end synapsis has been largely advanced through recent structural and single-molecule studies of NHEJ proteins. This review focuses on core NHEJ proteins that mediate DNA end synapsis through their unique structures and interaction properties, as well as how they play roles as anchor and linker proteins during the process of 'bridge over troubled ends'.
Topics: Chromosome Pairing; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA-Binding Proteins; Humans; Nucleic Acid Conformation
PubMed: 31829407
DOI: 10.1042/BST20180518 -
DNA Repair Oct 2021The nonhomologous DNA end joining pathway is required for repair of most double-strand breaks in the mammalian genome. Here we use a purified biochemical NHEJ system to...
The nonhomologous DNA end joining pathway is required for repair of most double-strand breaks in the mammalian genome. Here we use a purified biochemical NHEJ system to compare the joining of free DNA with recombinant mononucleosomal and dinucleosomal substrates to investigate ligation and local DNA end resection. We find that the nucleosomal state permits ligation in a manner dependent on the presence of free DNA flanking the nucleosome core particle. Local resection at DNA ends by the Artemis:DNA-PKcs nuclease complex is completely suppressed in all mononucleosome substrates regardless of flanking DNA up to a length of 14 bp. Like mononucleosomes, dinucleosomes lacking flanking free DNA are not joined. Therefore, the nucleosomal state imposes severe constraints on NHEJ nuclease and ligase activities.
Topics: Animals; Cell Line; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligases; DNA-Activated Protein Kinase; HeLa Cells; Humans; Nucleosomes; Spodoptera; Xenopus
PubMed: 34339948
DOI: 10.1016/j.dnarep.2021.103193 -
Molecules (Basel, Switzerland) May 2023Extrachromosomal circular DNA (eccDNA) is a special class of circular DNA in eukaryotes. Recent studies have suggested that eccDNA is the product of genomic instability...
Extrachromosomal circular DNA (eccDNA) is a special class of circular DNA in eukaryotes. Recent studies have suggested that eccDNA is the product of genomic instability and has important biological functions to regulate many downstream biological processes. While NGS (Next-Generation Sequencing)-based eccDNA sequencing has led to the identification of many eccDNAs in both healthy and diseased tissues, the specific biological functions of individual eccDNAs have yet to be clearly elucidated. Synthesizing eccDNAs longer than 1 kb with specific sequences remains a major challenge in the field, which has hindered our ability to fully understand their functions. Current methods for synthesizing eccDNAs primarily rely on chemical oligo synthesis, ligation, or the use of a specific gene editing and recombination systems. Therefore, these methods are often limited by the length of eccDNAs and are complex, expensive, as well as time-consuming. In this study, we introduce a novel method named QuickLAMA (Ligase-Assisted Minicircle Accumulation) for rapidly synthesizing eccDNAs up to 2.6 kb using a simple PCR and ligation approach. To validate the efficacy of our method, we synthesized three eccDNAs of varying lengths from cancer tissue and PC3 cells and confirmed successful circularization through sequencing and restriction enzyme digestion. Additional analyses have demonstrated that this method is highly efficient, cost-effective, and time-efficient, with good reproducibility. Using the method, a well-trained molecular biologist can synthesize and purify multiple eccDNAs within a single day, and it can be easily standardized and processed in a high-throughput manner, indicating the potential of the method to produce a wide range of desired eccDNAs and promote the translation of eccDNA research into clinical applications.
Topics: Reproducibility of Results; DNA, Circular; Polymerase Chain Reaction
PubMed: 37241975
DOI: 10.3390/molecules28104236 -
International Journal of Molecular... Feb 2023DNA topoisomerases have an essential role in resolving topological problems that arise due to the double-helical structure of DNA. They can recognise DNA topology and... (Review)
Review
DNA topoisomerases have an essential role in resolving topological problems that arise due to the double-helical structure of DNA. They can recognise DNA topology and catalyse diverse topological reactions by cutting and re-joining DNA ends. Type IA and IIA topoisomerases, which work by strand passage mechanisms, share catalytic domains for DNA binding and cleavage. Structural information has accumulated over the past decades, shedding light on the mechanisms of DNA cleavage and re-ligation. However, the structural rearrangements required for DNA-gate opening and strand transfer remain elusive, in particular for the type IA topoisomerases. In this review, we compare the structural similarities between the type IIA and type IA topoisomerases. The conformational changes that lead to the opening of the DNA-gate and strand passage, as well as allosteric regulation, are discussed, with a focus on the remaining questions about the mechanism of type IA topoisomerases.
Topics: DNA Topoisomerases; DNA; Isomerases; Catalytic Domain; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II
PubMed: 36835394
DOI: 10.3390/ijms24043986 -
BMC Genomics Dec 2019Cell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression....
BACKGROUND
Cell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression. Originally developed for the genomic analysis of highly degraded ancient DNA, single-stranded DNA (ssDNA) library preparation methods are gaining popularity in the field of cfDNA analysis due to their efficiency and ability to convert short, fragmented DNA into sequencing libraries without altering DNA ends. However, current ssDNA methods are costly and time-consuming.
RESULTS
Here we present an efficient ligation-based single-stranded library preparation method that is engineered to produce complex libraries in under 2.5 h from as little as 1 nanogram of input DNA without alteration to the native ends of template molecules. Our method, called Single Reaction Single-stranded LibrarY or SRSLY, ligates uniquely designed Next-Generation Sequencing (NGS) adapters in a one-step combined phosphorylation/ligation reaction that foregoes end-polishing. Using synthetic DNA oligos and cfDNA, we demonstrate the efficiency and utility of this approach and compare with existing double-stranded and single-stranded approaches for library generation. Finally, we demonstrate that cfDNA NGS data generated from SRSLY can be used to analyze DNA fragmentation patterns to deduce nucleosome positioning and transcription factor binding.
CONCLUSIONS
SRSLY is a versatile tool for converting short and fragmented DNA molecules, like cfDNA fragments, into sequencing libraries while retaining native lengths and ends.
Topics: Cell-Free Nucleic Acids; DNA, Single-Stranded; Gene Library; High-Throughput Nucleotide Sequencing; Humans; Oligonucleotides; Sequence Analysis, DNA
PubMed: 31881841
DOI: 10.1186/s12864-019-6355-0 -
Structure (London, England : 1993) Mar 2022In this issue of Structure, Sverzhinsky et al. (2022) report structures of archaeal DNA ligase bound to the proliferating cell nuclear antigen (PCNA) sliding clamp and...
In this issue of Structure, Sverzhinsky et al. (2022) report structures of archaeal DNA ligase bound to the proliferating cell nuclear antigen (PCNA) sliding clamp and a nicked DNA substrate. The structures provide snapshots of ligation intermediates, which reveal a dynamic nature of the complex and explain how PCNA stimulates the DNA ligase activity.
Topics: Cryoelectron Microscopy; DNA; DNA Ligase ATP; DNA Ligases; Proliferating Cell Nuclear Antigen; Protein Binding
PubMed: 35245433
DOI: 10.1016/j.str.2022.02.008 -
Nucleic Acids Research Jun 2023UV-damaged DNA-binding protein (UV-DDB) is a heterodimeric protein, consisting of DDB1 and DDB2 subunits, that works to recognize DNA lesions induced by UV damage during...
UV-damaged DNA-binding protein (UV-DDB) is a heterodimeric protein, consisting of DDB1 and DDB2 subunits, that works to recognize DNA lesions induced by UV damage during global genome nucleotide excision repair (GG-NER). Our laboratory previously discovered a non-canonical role for UV-DDB in the processing of 8-oxoG, by stimulating 8-oxoG glycosylase, OGG1, activity 3-fold, MUTYH activity 4-5-fold, and APE1 (apurinic/apyrimidinic endonuclease 1) activity 8-fold. 5-hydroxymethyl-deoxyuridine (5-hmdU) is an important oxidation product of thymidine which is removed by single-strand selective monofunctional DNA glycosylase (SMUG1). Biochemical experiments with purified proteins indicated that UV-DDB stimulates the excision activity of SMUG1 on several substrates by 4-5-fold. Electrophoretic mobility shift assays indicated that UV-DDB displaced SMUG1 from abasic site products. Single-molecule analysis revealed that UV-DDB decreases the half-life of SMUG1 on DNA by ∼8-fold. Immunofluorescence experiments demonstrated that cellular treatment with 5-hmdU (5 μM for 15 min), which is incorporated into DNA during replication, produces discrete foci of DDB2-mCherry, which co-localize with SMUG1-GFP. Proximity ligation assays supported a transient interaction between SMUG1 and DDB2 in cells. Poly(ADP)-ribose accumulated after 5-hmdU treatment, which was abrogated with SMUG1 and DDB2 knockdown. These data support a novel role for UV-DDB in the processing of the oxidized base, 5-hmdU.
Topics: DNA Damage; DNA-Binding Proteins; DNA Repair; DNA; Thymidine; Ultraviolet Rays
PubMed: 36971122
DOI: 10.1093/nar/gkad206