Enrichment of non-B-form DNA at D. melanogaster centromeres

Centromeres are essential chromosomal regions that mediate the accurate inheritance of genetic information during eukaryotic cell division. Despite their conserved function, centromeres do not contain conserved DNA sequences and are instead epigenetically marked by the presence of the centromere-specific histone H3 variant CENP-A (centromeric protein A). The functional contribution of centromeric DNA sequences to centromere identity remains elusive. Previous work found that dyad symmetries with a propensity to adopt non-canonical secondary DNA structures are enriched at the centromeres of several species. These findings lead to the proposal that such non-canonical DNA secondary structures may contribute to centromere specification. Here, we analyze the predicted secondary structures of the recently identified centromere DNA sequences from Drosophila melanogaster. Although dyad symmetries are only enriched on the Y centromere, we find that other types of non-canonical DNA structures, including DNA melting and G-quadruplexes, are common features of all D. melanogaster centromeres. Our work is consistent with previous models suggesting that non-canonical DNA secondary structures may be conserved features of centromeres with possible implications for centromere specification.

Non-B DNA Secondary Structures and Their Resolution by RecQ Helicases

Journal of Nucleic Acids ◽

10.4061/2011/724215 ◽

2011 ◽

Vol 2011 ◽

pp. 1-15 ◽

Cited By ~ 15

Author(s):

Sudha Sharma

Keyword(s):

Transcriptional Control ◽

Molecular Targets ◽

Secondary Structures ◽

Special Focus ◽

Dna Helicases ◽

Dna Structures ◽

Recq Helicases ◽

Alternative Structures ◽

In addition to the canonical B-form structure first described by Watson and Crick, DNA can adopt a number of alternative structures. These non-B-form DNA secondary structures form spontaneously on tracts of repeat sequences that are abundant in genomes. In addition, structured forms of DNA with intrastrand pairing may arise on single-stranded DNA produced transiently during various cellular processes. Such secondary structures have a range of biological functions but also induce genetic instability. Increasing evidence suggests that genomic instabilities induced by non-B DNA secondary structures result in predisposition to diseases. Secondary DNA structures also represent a new class of molecular targets for DNA-interactive compounds that might be useful for targeting telomeres and transcriptional control. The equilibrium between the duplex DNA and formation of multistranded non-B-form structures is partly dependent upon the helicases that unwind (resolve) these alternate DNA structures. With special focus on tetraplex, triplex, and cruciform, this paper summarizes the incidence of non-B DNA structures and their association with genomic instability and emphasizes the roles of RecQ-like DNA helicases in genome maintenance by resolution of DNA secondary structures. In future, RecQ helicases are anticipated to be additional molecular targets for cancer chemotherapeutics.

Inverted duplicate DNA sequences increase translocation rates through sequencing nanopores resulting in reduced base calling accuracy

Nucleic Acids Research ◽

10.1093/nar/gkaa206 ◽

2020 ◽

Vol 48 (9) ◽

pp. 4940-4945

Author(s):

Pieter Spealman ◽

Jaden Burrell ◽

David Gresham

Keyword(s):

Dna Sequences ◽

Copy Number ◽

Copy Number Variants ◽

Electrical Current ◽

Nanopore Sequencing ◽

Aberrant Behavior ◽

Structural Variants ◽

Dna Structures ◽

Base Calling ◽

Abstract Inverted duplicated DNA sequences are a common feature of structural variants (SVs) and copy number variants (CNVs). Analysis of CNVs containing inverted duplicated DNA sequences using nanopore sequencing identified recurrent aberrant behavior characterized by low confidence, incorrect and missed base calls. Inverted duplicate DNA sequences in both yeast and human samples were observed to have systematic elevation in the electrical current detected at the nanopore, increased translocation rates and decreased sampling rates. The coincidence of inverted duplicated DNA sequences with dramatically reduced sequencing accuracy and an increased translocation rate suggests that secondary DNA structures may interfere with the dynamics of transit of the DNA through the nanopore.

N-Terminus Does Not Govern Protein Turnover of Schizosaccharomyces pombe CENP-A

International Journal of Molecular Sciences ◽

10.3390/ijms21176175 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6175

Author(s):

Hwei Ling Tan ◽

Yi Bing Zeng ◽

Ee Sin Chen

Keyword(s):

Schizosaccharomyces Pombe ◽

Dna Sequences ◽

Protein Turnover ◽

Protein A ◽

Histone H3 ◽

Epigenetic Inheritance ◽

Cell Extract ◽

Insoluble Fraction ◽

Independent Manner ◽

Histone H3 Variant

Centromere integrity underlies an essential framework for precise chromosome segregation and epigenetic inheritance. Although centromeric DNA sequences vary among different organisms, all eukaryotic centromeres comprise a centromere-specific histone H3 variant, centromeric protein A (CENP-A), on which other centromeric proteins assemble into the kinetochore complex. This complex connects chromosomes to mitotic spindle microtubules to ensure accurate partitioning of the genome into daughter cells. Overexpression of CENP-A is associated with many cancers and is correlated with its mistargeting, forming extra-centromeric kinetochore structures. The mislocalization of CENP-A can be counteracted by proteolysis. The amino (N)-terminal domain (NTD) of CENP-A has been implicated in this regulation and shown to be dependent on the proline residues within this domain in Saccharomyces cerevisiae CENP-A, Cse4. We recently identified a proline-rich GRANT motif in the NTD of Schizosaccharomyces pombe CENP-A (SpCENP-A) that regulates the centromeric targeting of CENP-A via binding to the CENP-A chaperone Sim3. Here, we investigated whether the NTD is required to confer SpCENP-A turnover (i.e., counter stability) using various truncation mutants of SpCENP-A. We show that sequential truncation of the NTD did not improve the stability of the protein, indicating that the NTD of SpCENP-A does not drive turnover of the protein. Instead, we reproduced previous observations that heterochromatin integrity is important for SpCENP-A stability, and showed that this occurs in an NTD-independent manner. Cells bearing the null mutant of the histone H3 lysine 9 methyltransferase Clr4 (Δclr4), which have compromised constitutive heterochromatin integrity, showed reductions in the proportion of SpCENP-A in the chromatin-containing insoluble fraction of the cell extract, suggesting that heterochromatin may promote SpCENP-A chromatin incorporation. Thus, a disruption in heterochromatin may result in the delocalization of SpCENP-A from chromatin, thus exposing it to protein turnover. Taken together, we show that the NTD is not required to confer SpCENP-A protein turnover.

Experimental Confirmation of Multiple Co-Existent DNA Secondary Structures using Low-Yield Bisulfite Sequencing

10.1101/2021.05.21.445174 ◽

2021 ◽

Author(s):

Jiaming Li ◽

Jin Bae ◽

Boyan Yordanov ◽

Michael X Wang ◽

Javier Gonzalez ◽

...

Keyword(s):

Secondary Structure ◽

Single Molecule ◽

Dna Sequences ◽

Bisulfite Sequencing ◽

Secondary Structures ◽

Single Molecule Level ◽

Dna Oligonucleotides ◽

Metastable Structures ◽

Structure State ◽

Dna Secondary Structures

The prediction of DNA secondary structures from DNA sequences using thermodynamic models is imperfect for many biological sequences, both due to insufficient experimental data for training and to the kinetics of folding that lead to metastable structures. Here, we developed low-yield bisulfite sequencing (LYB-seq) to query the secondary structure states of cytosine (C) nucleotides in thousands of different DNA oligonucleotides on a single-molecule level. We observed that the reaction kinetics between bisulfite and C nucleotides is highly dependent on the secondary structure state of the C nucleotides, with the most accessible C nucleotides (those in small hairpin loops) reacting 70-fold faster than those in stable duplexes. Next, we developed a statistical model to evaluate the likelihood of an NGS read being consistent with a particular proposed secondary structure. By analyzing thousands of NGS reads for each DNA species, we can infer the distribution of secondary structures adopted by each species in solution. We find that 84% of 1,057 human genome subsequences studied here adopt 2 or more stable secondary structures in solution.

A long non-coding RNA is required for targeting centromeric protein A to the human centromere

eLife ◽

10.7554/elife.03254 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 106

Author(s):

Delphine Quénet ◽

Yamini Dalal

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Biochemical Characterization ◽

Protein A ◽

Human Centromere ◽

Non Coding Rna ◽

Histone H3 Variant ◽

Centromeric Protein ◽

Long Non Coding Rna

The centromere is a specialized chromatin region marked by the histone H3 variant CENP-A. Although active centromeric transcription has been documented for over a decade, the role of centromeric transcription or transcripts has been elusive. Here, we report that centromeric α-satellite transcription is dependent on RNA Polymerase II and occurs at late mitosis into early G1, concurrent with the timing of new CENP-A assembly. Inhibition of RNA Polymerase II-dependent transcription abrogates the recruitment of CENP-A and its chaperone HJURP to native human centromeres. Biochemical characterization of CENP-A associated RNAs reveals a 1.3 kb molecule that originates from centromeres, which physically interacts with the soluble pre-assembly HJURP/CENP-A complex in vivo, and whose down-regulation leads to the loss of CENP-A and HJURP at centromeres. This study describes a novel function for human centromeric long non-coding RNAs in the recruitment of HJURP and CENP-A, implicating RNA-based chaperone targeting in histone variant assembly.

Replication of G Quadruplex DNA

Genes ◽

10.3390/genes10020095 ◽

2019 ◽

Vol 10 (2) ◽

pp. 95 ◽

Cited By ~ 34

Author(s):

Leticia Koch Lerner ◽

Julian E. Sale

Keyword(s):

Dna Replication ◽

Dna Sequences ◽

Secondary Structures ◽

Dna Unwinding ◽

Chromosomal Dna ◽

Quadruplex Dna ◽

Epigenetic Instability ◽

G Quadruplex ◽

Dna Template

A cursory look at any textbook image of DNA replication might suggest that the complex machine that is the replisome runs smoothly along the chromosomal DNA. However, many DNA sequences can adopt non-B form secondary structures and these have the potential to impede progression of the replisome. A picture is emerging in which the maintenance of processive DNA replication requires the action of a significant number of additional proteins beyond the core replisome to resolve secondary structures in the DNA template. By ensuring that DNA synthesis remains closely coupled to DNA unwinding by the replicative helicase, these factors prevent impediments to the replisome from causing genetic and epigenetic instability. This review considers the circumstances in which DNA forms secondary structures, the potential responses of the eukaryotic replisome to these impediments in the light of recent advances in our understanding of its structure and operation and the mechanisms cells deploy to remove secondary structure from the DNA. To illustrate the principles involved, we focus on one of the best understood DNA secondary structures, G quadruplexes (G4s), and on the helicases that promote their resolution.

Nanopore sequencing undergoes catastrophic sequence failure at inverted duplicated DNA sequences

10.1101/852665 ◽

2019 ◽

Cited By ~ 2

Author(s):

Pieter Spealman ◽

Jaden Burrell ◽

David Gresham

Keyword(s):

Dna Sequences ◽

Copy Number ◽

Copy Number Variants ◽

Nanopore Sequencing ◽

Aberrant Behavior ◽

Structural Variants ◽

Dna Structures ◽

Inverted duplicated sequences are a common feature of structural variants (SVs) and copy number variants (CNVs). Analysis of CNVs containing inverted duplicated sequences using nanopore sequencing identified recurrent aberrant behavior characterized by incorrect and low confidence base calls that result from a systematic elevation in the current recorded by the sequencing pore. The coincidence of inverted duplicated sequences with catastrophic sequence failure suggests that secondary DNA structures may impair transit through the nanopore.

HERC2 regulates RPA2 by mediating ATR-induced Ser33 phosphorylation and ubiquitin-dependent degradation

Scientific Reports ◽

10.1038/s41598-019-50812-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Yongqiang Lai ◽

Mingzhang Zhu ◽

Wenwen Wu ◽

Nana Rokutanda ◽

Yukiko Togashi ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Genome Stability ◽

Protein A ◽

E3 Ubiquitin Ligase ◽

Hect Domain ◽

Dna Structures ◽

G4 Dna ◽

G Quadruplex ◽

Secondary Dna Structures ◽

Sirna Knockdown

Abstract Replication protein A (RPA) binds to and stabilizes single-stranded DNA and is essential for the genome stability. We reported that an E3 ubiquitin ligase, HERC2, suppresses G-quadruplex (G4) DNA by regulating RPA-helicase complexes. However, the precise mechanism of HERC2 on RPA is as yet largely unknown. Here, we show essential roles for HERC2 on RPA2 status: induction of phosphorylation and degradation of the modified form. HERC2 interacted with RPA through the C-terminal HECT domain. Ubiquitination of RPA2 was inhibited by HERC2 depletion and rescued by reintroduction of the C-terminal fragment of HERC2. ATR-mediated phosphorylation of RPA2 at Ser33 induced by low-level replication stress was inhibited by depletion of HERC2. Contrary, cells lacking HERC2 catalytic residues constitutively expressed an increased level of Ser33-phosphorylated RPA2. HERC2-mediated ubiquitination of RPA2 was abolished by an ATR inhibitor, supporting a hypothesis that the ubiquitinated RPA2 is a phosphorylated subset. Functionally, HERC2 E3 activity has an epistatic relationship with RPA in the suppression of G4 when judged with siRNA knockdown experiments. Together, these results suggest that HERC2 fine-tunes ATR-phosphorylated RPA2 levels through induction and degradation, a mechanism that could be critical for the suppression of secondary DNA structures during cell proliferation.

5′ to 3′ Unfolding Directionality of DNA Secondary Structures by Replication Protein A

Journal of Biological Chemistry ◽

10.1074/jbc.m115.709667 ◽

2016 ◽

Vol 291 (40) ◽

pp. 21246-21256 ◽

Cited By ~ 22

Author(s):

Layal Safa ◽

Nassima Meriem Gueddouda ◽

Frédéric Thiébaut ◽

Emmanuelle Delagoutte ◽

Irina Petruseva ◽

...

Keyword(s):

Protein A ◽

Replication Protein A ◽

Secondary Structures ◽

Replication Protein ◽

Dna Secondary Structures

DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

Nucleic Acids Research ◽

10.1093/nar/gkt1174 ◽

2013 ◽

Vol 42 (4) ◽

pp. 2270-2281 ◽

Cited By ~ 31

Author(s):

Adam F. Sander ◽

Thomas Lavstsen ◽

Thomas S. Rask ◽

Michael Lisby ◽

Ali Salanti ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Membrane Protein ◽

Erythrocyte Membrane ◽

Gene Families ◽

Secondary Structures ◽

Erythrocyte Membrane Protein ◽

Sexual Stages ◽

Falciparum Erythrocyte Membrane Protein

Parasitic Pathogens ◽

Abstract Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens.