The Incorporation of Ribonucleotides Induces Structural and Conformational Changes in DNA

Abstract

Ribonucleotide incorporation is the most common error occurring during DNA replication. Cells have hence developed mechanisms to remove ribonucleotides from the genome and restore its integrity. Indeed, the persistence of ribonucleotides into DNA leads to severe consequences, such as genome instability and replication stress. Thus, it becomes important to understand the effects of ribonucleotides incorporation, starting from their impact on DNA structure and conformation. Here we present a systematic study of the effects of ribonucleotide incorporation into DNA molecules. We have developed, to our knowledge, a new method to efficiently synthesize long DNA molecules (hundreds of basepairs) containing ribonucleotides, which is based on a modified protocol for the polymerase chain reaction. By means of atomic force microscopy, we could therefore investigate the changes, upon ribonucleotide incorporation, of the structural and conformational properties of numerous DNA populations at the single-molecule level. Specifically, we characterized the scaling of the contour length with the number of basepairs and the scaling of the end-to-end distance with the curvilinear distance, the bending angle distribution, and the persistence length. Our results revealed that ribonucleotides affect DNA structure and conformation on scales that go well beyond the typical dimension of the single ribonucleotide. In particular, the presence of ribonucleotides induces a systematic shortening of the molecules, together with a decrease of the persistence length. Such structural changes are also likely to occur in vivo, where they could directly affect the downstream DNA transactions, as well as interfere with protein binding and recognition.

New perspectives in cancer biology from a study of canonical and non-canonical functions of base excision repair proteins with a focus on early steps

“…The biological relevance of rNMP incorporation in genomic DNA is emerging from a growing body of recent scientific studies. Whatever the cause of the rNMP(s) incorporation, the additional 2′-OH group on one or more rNMP(s) alters DNA elasticity and structure in a sequence-dependent manner, destabilising the DNA backbone, increasing the susceptibility to DNA hydrolysis, and finally causing strand cleavage and/or mutability (148,(169)(170)(171)(172)(173)(174). The genomic DNA stability, perturbed by the rNMP(s) presence, is preserved by the combined action of several enzymes orchestrated in a unique pathway called ribonucleotide excision repair (RER) (160).…”

Molecular and physiological consequences of faulty eukaryotic ribonucleotide excision repair

“…The EMBO Journal 39: e102309 | 2020 ª 2019 The Authors containing DNA molecules (Chiu et al, 2014). Similar AFM analysis employing DNA molecules of up to one kilobase in length (i.e., more closely mimicking the in vivo context of rNMP sites) containing randomly incorporated rCTPs demonstrated shortening and increased elasticity of the DNA backbone (Meroni et al, 2017), suggesting that rNMPs can profoundly impact on the conformation of large DNA molecules beyond just their sites of incorporation. Albeit difficult to prove experimentally, it is conceivable that these structural effects of rNMPs on the surrounding DNA may play an important role to enhance their recognition and subsequent removal.…”

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].…”

“…Additionally, multiple rNMPs in the nucleus of S. cerevisiae cells are only tolerated, thanks to the action of the two main pathways of PRR: template-switch and TLS pol ζ [66]. Finally, similarly to single rNMPs, but even more significantly, polyribonucleotide chains may alter the proper conformation of DNA [59,60,62] and interfere with protein binding [63,64], possibly causing catastrophic defects in chromosome segregation and a global alteration of gene expression profiles. For all these reasons, further investigation of multiple rNMPs' metabolism appears very important.…”