Rotational and translational positions determine the structural and dynamic impact of a single ribonucleotide incorporated in the nucleosome

Abstract

Ribonucleotides misincorporated by replicative DNA polymerases are by far the most common DNA lesion. The presence of ribonucleotides in DNA is associated with genome instability, causing replication stress, chromosome fragility, gross chromosomal rearrangements, and other mutagenic events. Furthermore, nucleosome and chromatin assembly as well as nucleosome positioning are affected by the presence of ribonucleotides. Notably, nucleosome formation is significantly reduced by a single ribonucleotide. Single ribonucleotides are primarily removed from DNA by the ribonucleotide excision repair (RER) pathway via the RNase H2 enzyme, which incises the DNA backbone on the 5’-side of the ribonucleotide. While the structural implications of a single ribonucleotide in free duplex DNA have been well studied, how a single ribonucleotide embedded in nucleosomal DNA impacts nucleosome structure and dynamics, and the possible consequent impact on RER, have not been explored. We have carried out 3.5 microsecond molecular dynamics simulations of a single ribonucleotide incorporated at various translational and rotational positions in a nucleosome core particle. We find that the presence of the 2’-OH group on the ribose impacts the local conformation and dynamics of both the ribonucleotide and nearby DNA nucleotides as well as their interactions with histones; the nature of these disturbances depends on the rotational and translational setting, including whether the ribose faces toward or away from the histones. The ribonucleotide’s preferred C3’-endo pucker is stabilized by interactions with the histones, and furthermore the ribonucleotide can cause dynamic local duplex disturbance involving an abnormal C3’-endo population of the adjacent deoxyribose pucker, minor groove opening, ruptured Watson-Crick pairing, and duplex unwinding that are governed by translation-dependent histone-nucleotide interactions. Possible effects of these disturbances on RER are considered.

Molecular and physiological consequences of faulty eukaryotic ribonucleotide excision repair

“…The consequential defects in nucleosome formation and stability could result in severe consequences on cellular fitness. In contrast, inward-facing rNMPs, especially at a translational position that directly interacts with the nucleosome, were mostly found in a stable C3 0 -endo conformation (associated with A-DNA) and consequently increased the width of the DNA minor grove, disrupted Watson-Crick base pairing, and induced local unwinding of the DNA duplex (Fu et al, 2018). Such global effects are likely to influence cellular processes that are regulated by the state of chromatin, including gene expression, chromosome segregation, and DNA repair (Fig 4).…”

“…Outward-facing rNMPs, independent of their translational position, adopted a C2 0endo conformation (associated with B-DNA) and did not affect adjacent base pairs, rendering them indistinguishable from dNMPs in terms of their overall structure. In contrast, inward-facing rNMPs, especially at a translational position that directly interacts with the nucleosome, were mostly found in a stable C3 0 -endo conformation (associated with A-DNA) and consequently increased the width of the DNA minor grove, disrupted Watson-Crick base pairing, and induced local unwinding of the DNA duplex (Fu et al, 2018). Besides demonstrating a direct effect of rNMPs on DNA structure, these findings also implicate that RER efficiency might depend on the position of the rNMP within the nucleosome.…”

“…In this regard, in vitro studies point toward a strong negative impact of RNA-containing DNA on nucleosome formation (Dunn & Griffith, 1980;Hovatter & Martinson, 1987). Recent molecular dynamics simulations addressing the direct effect of ribonucleotides in the context of the nucleosome have modeled a single rNMP at different positions within a DNA-bound histone octamer (Fu et al, 2018). Less stable nucleosomes may cause altered occupation patterns, decreased nucleosome density, and consequently a less densely packed genome.…”

“…Such global effects are likely to influence cellular processes that are regulated by the state of chromatin, including gene expression, chromosome segregation, and DNA repair (Fig 4). Recent molecular dynamics simulations addressing the direct effect of ribonucleotides in the context of the nucleosome have modeled a single rNMP at different positions within a DNA-bound histone octamer (Fu et al, 2018). Interestingly, nucleosome formation was differentially affected by both the translational setting (distance from nucleosome midpoint) and the rotational setting (facing outwards or toward the nucleosome) of the rNMP.…”

One, No One, and One Hundred Thousand: The Many Forms of Ribonucleotides in DNA

“…Single chromosome-embedded rNMPs must be anyway promptly removed, as their persistence has several negative consequences. Ribonucleotides left in DNA alter the shape and the conformation of DNA molecules [59][60][61][62], the assembly of nucleosomes [63,64], and they may hamper DNA replication since replicative DNA polymerases ε and δ are not efficient in bypassing them [16,18,19,[65][66][67][68]. However, the most detrimental effects of single rNMPs seem to derive from their improper repair, as reviewed in [3].…”