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.

Restarted replication forks are error-prone and cause CAG repeat expansions and contractions

Disease-associated trinucleotide repeats form secondary DNA structures that interfere with replication and repair. Replication has been implicated as a mechanism that can cause repeat expansions and contractions. However, because structure-forming repeats are also replication barriers, it has been unclear whether the instability occurs due to slippage during normal replication progression through the repeat, slippage or misalignment at a replication stall caused by the repeat, or during subsequent replication of the repeat by a restarted fork that has altered properties. In this study, we have specifically addressed the fidelity of a restarted fork as it replicates through a CAG/CTG repeat tract and its effect on repeat instability. To do this, we used a well-characterized site-specific replication fork barrier (RFB) system in fission yeast that creates an inducible and highly efficient stall that is known to restart by recombination-dependent replication (RDR), in combination with long CAG repeat tracts inserted at various distances and orientations with respect to the RFB. We find that replication by the restarted fork exhibits low fidelity through repeat sequences placed 2–7 kb from the RFB, exhibiting elevated levels of Rad52- and Rad8ScRad5/HsHLTF-dependent instability. CAG expansions and contractions are not elevated to the same degree when the tract is just in front or behind the barrier, suggesting that the long-traveling Polδ-Polδ restarted fork, rather than fork reversal or initial D-loop synthesis through the repeat during stalling and restart, is the greatest source of repeat instability. The switch in replication direction that occurs due to replication from a converging fork while the stalled fork is held at the barrier is also a significant contributor to the repeat instability profile. Our results shed light on a long-standing question of how fork stalling and RDR contribute to expansions and contractions of structure-forming trinucleotide repeats, and reveal that tolerance to replication stress by fork restart comes at the cost of increased instability of repetitive sequences.

Centromeres under Pressure: Evolutionary Innovation in Conflict with Conserved Function

Centromeres are essential genetic elements that enable spindle microtubule attachment for chromosome segregation during mitosis and meiosis. While this function is preserved across species, centromeres display an array of dynamic features, including: (1) rapidly evolving DNA; (2) wide evolutionary diversity in size, shape and organization; (3) evidence of mutational processes to generate homogenized repetitive arrays that characterize centromeres in several species; (4) tolerance to changes in position, as in the case of neocentromeres; and (5) intrinsic fragility derived by sequence composition and secondary DNA structures. Centromere drive underlies rapid centromere DNA evolution due to the “selfish” pursuit to bias meiotic transmission and promote the propagation of stronger centromeres. Yet, the origins of other dynamic features of centromeres remain unclear. Here, we review our current understanding of centromere evolution and plasticity. We also detail the mutagenic processes proposed to shape the divergent genetic nature of centromeres. Changes to centromeres are not simply evolutionary relics, but ongoing shifts that on one side promote centromere flexibility, but on the other can undermine centromere integrity and function with potential pathological implications such as genome instability.

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

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.

Nanopore sequencing undergoes catastrophic sequence failure at inverted duplicated DNA sequences

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

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.

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

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.

Secondary DNA structures as molecular targets for cancer therapeutics

DNA sequence information is pivotal to transcription, replication and recombination. DNA structure is dependent upon intracellular conditions such as ion concentration and the presence of proteins that may bind to DNA to facilitate the interconversion between different forms and to stabilize specific secondary structures. Dependent upon the primary DNA sequence, purine- and pyrimidine-rich strands of DNA can adopt four-stranded structures known as G-quadruplexes and i-motifs, respectively. These structures have been proposed to exist in biologically important regions of DNA, e.g. at the end of chromosomes and in the regulatory regions of oncogenes such as c-myc. Proteins such as topoisomerase I and Rap1 can facilitate the formation of G-quadruplex structures, and for transcriptional activation of c-myc, proteins such as NM23–H2 and hnRNP K are required. These proteins bind to the non-duplex forms of the nuclease hypersensitivity element III, of c-myc. The design and synthesis of small molecules that target these secondary DNA structures and the biochemical and biological effects of these compounds are of potential importance in cancer chemotherapy.