Non-B DNA: a major contributor to small- and large-scale variation in nucleotide substitution frequencies across the genome

Abstract

Approximately 13% of the human genome can fold into non-canonical (non-B) DNA structures (e.g. G-quadruplexes, Z-DNA, etc.), which have been implicated in vital cellular processes. Non-B DNA also hinders replication, increasing errors and facilitating mutagenesis, yet its contribution to genome-wide variation in mutation rates remains unexplored. Here, we conducted a comprehensive analysis of nucleotide substitution frequencies at non-B DNA loci within noncoding, non-repetitive genome regions, their ±2 kb flanking regions, and 1-Megabase windows, using human-orangutan divergence and human single-nucleotide polymorphisms. Functional data analysis at single-base resolution demonstrated that substitution frequencies are usually elevated at non-B DNA, with patterns specific to each non-B DNA type. Mirror, direct and inverted repeats have higher substitution frequencies in spacers than in repeat arms, whereas G-quadruplexes, particularly stable ones, have higher substitution frequencies in loops than in stems. Several non-B DNA types also affect substitution frequencies in their flanking regions. Finally, non-B DNA explains more variation than any other predictor in multiple regression models for diversity or divergence at 1-Megabase scale. Thus, non-B DNA substantially contributes to variation in substitution frequencies at small and large scales. Our results highlight the role of non-B DNA in germline mutagenesis with implications to evolution and genetic diseases.

ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress

“…It has been shown that G4 structures are correlated with increased DNA polymerization error rates 49 , and that G4 structures harbor a high incidence of disease-causing point mutations and indels [50][51][52] . Further, studies demonstrate that ATRX mutant tumors carry a high mutation rate either at the single-nucleotide variant level or copy number alterations 25,53 .…”

Selection and thermostability suggest G-quadruplexes are novel functional elements of the human genome

“…Thus, selective constraints might be more relaxed at G4 versus non-G4 sites in some functional regions, leading to the observed trends. Alternatively, but not exclusively, the increase in polymorphic rate at G4s might be owing to their elevated mutation rate (Guiblet et al 2021).…”

“…If stable G4s are functional, then they are expected to be more conserved (i.e., to have lower human-orangutan divergence) than unstable G4s or non-G4 sites. However, elevated mutation rates at G4s, and at stable G4s in particular (Guiblet et al 2021), might also affect divergence. To analyze this effect on divergence, we initially focused on the putatively neutral NCNR, for which selection effects should be minimal.…”

“…G4 structures could act as a doubleedged sword for the genome. Whereas some G4 structures perform important functions, many G4 structures have the potential to hinder nucleic acid polymerization during replication, transcription, and translation (for review, see Varshney et al 2020) and to lead to increases in germline (Du et al 2014;Guiblet et al 2021) and somatic (Georgakopoulos-Soares et al 2018) mutations as well as to genomic instability (Cheung et al 2002;Zhao et al 2010). Thus, G4 structures that are not functional might be detrimental to the genome and hence might be selected against (Valton and Prioleau 2016).…”

“…Subsequently, a machine-learning model trained on these relative stability estimates and validated on 392 experimentally studied G4 structures was developed as the software tool Quadron, which is used to annotate G4 loci and predict their stability across the human genome (Sahakyan et al 2017a). Considering stability in studies of G4s is critical because (1) more stable G4s are known to have higher mutability (Guiblet et al 2021), and (2) we expect G4 stability to affect their function. Capitalizing on these recent developments, we investigated stability and selection among G4 loci harbored by different genic components (e.g., promoters, UTRs, and protein-coding exons) and nongenic functional regions (e.g., replication origins and enhancers) in the human genome.…”