The parallel-stranded d(CGA) duplex is a highly predictable structural motif with two conformationally distinct strands

ABSTRACTDNA can adopt non-canonical structures that have important biological functions while also providing structural diversity for nanotechnology applications. Here, we describe the crystal structures of two oligonucleotides composed of d(CGA) triplet repeats in the parallel-stranded duplex form. The structure determination of four unique d(CGA)-based parallel-stranded duplexes across two crystal structures has allowed us to characterize and establish structural parameters of d(CGA) triplets in the parallel-stranded duplex form. Our results show that d(CGA) units are highly uniform, but that each strand in the duplex is structurally unique and has a distinct role in accommodating structural asymmetries induced by the C-CH+ base pair.

What can go wrong in the non-coding genome and how to interpret whole genome sequencing data

Whole exome sequencing discovers causative mutations in less than 50 % of rare disease patients, suggesting the presence of additional mutations in the non-coding genome. So far, non-coding mutations have been identified in less than 0.2 % of individuals with genetic diseases listed in the ClinVar database and exhibit highly diverse molecular mechanisms. In contrast to our capability to sequence the whole genome, our ability to discover and functionally confirm such non-coding mutations is lagging behind severely. We discuss the problems and present examples of confirmed mutations in deep intronic sequences, non-coding triplet repeats, enhancers, and larger structural variants and highlight their proposed disease mechanisms. Finally, we discuss the type of data that would be required to establish non-coding mutation detection in routine diagnostics.

Development and validation in 500 female samples of a TP-PCR assay to identify AFF2 GCC expansions

The absence of FRAXE pathognomonic features hampers early recognition, delaying testing and molecular confirmation. Hence, our laboratory uses a multiplex PCR-based strategy to determine the allele size of both FRAXA and FRAXE in which around 20% of female samples are uninformative. The aim of this study is to develop a timely triplet-primed amplification (TP-PCR) screening strategy to size the AFF2 GCC repeat and accurately assess homozygosity in female samples. In order to achieve this, validation was performed in a cohort of 500 females with a previous uninformative FRAXE PCR result. Unlike the presence of AGG interspersion(s) in FRAXA triplet repeats, no interspersion was observed among the samples tested. Interestingly, the presence of a T>C SNP (rs868949662), contiguous to the GCC repetitive track, allows triplet primer binding in two additional GCCs, increasing the discrimination power of the TP-PCR assay in heterozygous and homozygous samples. Twelve alleles outside the normal range, eight intermediate and four premutated, were recognized, which seems relevant considering the rarity of the AFF2 expansions. Overall, this study confirmed the absence of interspersions in both normal and expanded FRAXE alleles, providing a strict, reproducible and low-cost homozygosity screening strategy. Furthermore, the occurrence of intermediate repeat sizes with unexpected frequency introduces new areas of clinical research in this cohort in understanding these less explored AFF2 repeat sizes and newly associated phenotypes.

The nuclear pore complex prevents sister chromatid recombination during replicative senescence

The Nuclear Pore Complex (NPC) has emerged as an important hub for processing various types of DNA damage. Here, we uncover that fusing a DNA binding domain to the NPC basket protein Nup1 reduces telomere relocalization to nuclear pores early after telomerase inactivation. This Nup1 modification also impairs the relocalization to the NPC of expanded CAG/CTG triplet repeats. Strikingly, telomerase negative cells bypass senescence when expressing this Nup1 modification by maintaining a minimal telomere length compatible with proliferation through rampant unequal exchanges between sister chromatids. We further report that a Nup1 mutant lacking 36 C-terminal residues recapitulates the phenotypes of the Nup1-LexA fusion indicating a direct role of Nup1 in the relocation of stalled forks to NPCs and restriction of error-prone recombination between repeated sequences. Our results reveal a new mode of telomere maintenance that could shed light on how 20% of cancer cells are maintained without telomerase or ALT.

Expansion of trinucleotide CTG repeats in the TCF4 gene as a marker of fuchs’ endothelial corneal dystrophy

Fuchs’ endothelial corneal dystrophy (FECD) is an inherited severe and progressive disease, characte­rized by endothelial cell density decrease and increasing corneal edema. FECD development may be linked to expanded trinucleotide repeat, CTG, in the third intron of the TCF4 gene. The study focuses on estimating the prevalence of expanded CTG repeat in TCF4 gene in the Russian population, in patients with normal cornea and in patients with FECD (by applying triplet repeat PCR technique and capillary electrophoresis). 51 patients with FECD and 38 patients with normal cornea were examined. The estimation of the number of CTG triplet repeats in TCF4 gene determination is the veracious laboratory marker of FECD.

Evolving Notch polyQ tracts reveal possible solenoid interference elements

ABSTRACTPolyglutamine (polyQ) tracts in regulatory proteins are extremely polymorphic. As functional elements under selection for length, triplet repeats are prone to DNA replication slippage and indel mutations. Many polyQ tracts are also embedded within intrinsically disordered domains, which are less constrained, fast evolving, and difficult to characterize. To identify structural principles underlying polyQ tracts in disordered regulatory domains, here I analyze deep evolution of metazoan Notch polyQ tracts, which can generate alleles causing developmental and neurogenic defects. I show that Notch features polyQ tract turnover that is restricted to a discrete number of conserved “polyQ insertion slots”. Notch polyQ insertion slots are: (i) identifiable by an amphipathic “slot leader” motif; (ii) conserved as an intact C-terminal array in a 1-to-1 relationship with the N-terminal solenoid-forming ankyrin repeats (ARs); and (iii) enriched in carboxamide residues (Q/N), whose sidechains feature dual hydrogen bond donor and acceptor atoms. Correspondingly, the terminal loop and β-strand of each AR feature conserved carboxamide residues, which would be susceptible to folding interference by hydrogen bonding with residues outside the ARs. I thus suggest that Notch polyQ insertion slots constitute an array of AR interference elements (ARIEs). Notch ARIEs would dynamically compete with the delicate serial folding induced by adjacent ARs. Huntingtin, which harbors solenoid-forming HEAT repeats, also possesses a similar number of polyQ insertion slots. These results strongly suggest that intrinsically disordered interference arrays featuring carboxamide and polyQ enrichment are coupled proteodynamic modulators of solenoids.SIGNIFICANCENeurodegenerative disorders are often caused by expanded polyglutamine (polyQ) tracts embedded in the disordered regions of regulatory proteins, which are difficult to characterize structurally. To identify functional principles underlying polyQ tracts in disordered regulatory domains, I analyze evolution of the Notch protein, which can generate polyQ-related alleles causing neurodevelopmental defects. I show that Notch evolves polyQ tracts that come and go in a few conserved “polyQ insertion slots”. Several features suggest these slots are ankyrin repeat (AR) interference elements, which dynamically compete with the delicate solenoid formed by Notch. Huntingtin, whose polyQ expansions causes Huntington’s Disease in humans, also has solenoid-forming modules and polyQ insertion slots, suggesting a common architectural principle underlies solenoid-forming polyQ-rich proteins.