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  • Review Article
  • Published:

Processing ribonucleotides incorporated during eukaryotic DNA replication

Nature Reviews Molecular Cell Biology volume 17, pages 350–363 (2016)Cite this article

Subjects

Key Points

  • Ribonucleotides are incorporated into DNA during replication.

  • Ribonucleotides can be removed during ribonucleotide excision repair or topoisomerase I-initiated processing.

  • Failure of ribonucleotide removal is associated with genome instability in the form of mutagenesis, replication stress, DNA breaks and chromosomal rearrangements.

  • Human diseases, including autoimmune disorders and neurodegenerative diseases, may be associated with failure to process genomic ribonucleotides

  • Genomic ribonucleotides function as a strand-discrimination signal during DNA mismatch repair, and they may have other physiological roles.

Abstract

The information encoded in DNA is influenced by the presence of non-canonical nucleotides, the most frequent of which are ribonucleotides. In this Review, we discuss recent discoveries about ribonucleotide incorporation into DNA during replication by the three major eukaryotic replicases, DNA polymerases α, δ and ε. The presence of ribonucleotides in DNA causes short deletion mutations and may result in the generation of single- and double-strand DNA breaks, leading to genome instability. We describe how these ribonucleotides are removed from DNA through ribonucleotide excision repair and by topoisomerase I. We discuss the biological consequences and the physiological roles of ribonucleotides in DNA, and consider how deficiencies in their removal from DNA may be important in the aetiology of disease.

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Figure 1: Ribonucleotide incorporation into DNA by polymerases.
Figure 2: Ribonucleotide removal during ribonucleotide excision repair or topoisomerase I processing.
Figure 3: Consequences of unrepaired ribonucleotides in DNA.
Figure 4: The connections between genomic ribonucleotides and human disease.
Figure 5: Ribonucleotides function as a strand-discrimination signal during DNA mismatch repair.

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Acknowledgements

The authors thank Dmitry Gordenin and Scott Williams for critical reading of the manuscript. The laboratory is supported by the Intramural Research Program of the US National Institutes of Health, National Institute of Environmental Health Sciences. The authors apologize to colleagues whose primary research articles are not cited owing to space limitations.

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  1. Department of Health and Human Services (DHHS), Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, 27709, North Carolina, USA

    Jessica S. Williams, Scott A. Lujan & Thomas A. Kunkel

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  1. Jessica S. Williams
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  2. Scott A. Lujan
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PowerPoint slides

Glossary

DNA polymerases

Enzymes that synthesize chains of deoxyribonucleotides.

Translesion DNA synthesis

(TLS). DNA synthesis that occurs across a template DNA lesion using a specialized DNA polymerase.

R-loops

Three-stranded structures that include both an RNA–DNA hybrid and a single DNA strand. R-loops are formed during transcription when the nascent mRNA hybridizes with the complementary template DNA strand and, if not removed, can threaten genome stability or regulate gene expression.

Topoisomerase I

(Top1). A type 1B topoisomerase that relieves both positive and negative DNA supercoils by generating a reversible single-strand DNA nick in duplex DNA. Top1 functions during both DNA replication and transcription.

Okazaki fragment

A short DNA fragment synthesized by DNA polymerase α (Pol α) and Pol δ with a length determined by nucleosome periodicity. Following removal of the RNA primer (synthesized by Pol α) to initiate synthesis, these segments are joined by DNA ligase I to form the continuous lagging strand.

Proofreading

The 3′–5′ exonuclease activity possessed by some DNA polymerases that facilitates the removal of DNA mismatches before synthesis continues.

Mismatch repair

(MMR). A post-replication repair process that removes base–base mismatches and deletions resulting from DNA synthesis errors. MMR occurs in a strand-specific manner and involves error recognition, excision and gap re-synthesis.

RNase H2

A conserved heterotrimeric enzyme that cleaves the RNA portion of a DNA–RNA hybrid, hydrolysing both single and multiple consecutive ribonucleotides in DNA.

B family DNA polymerases

A family of related DNA polymerases that are found in all domains of cellular life. The family includes the highly accurate eukaryotic and archaeal replicases, and the polymerases from phages T4 and RB69. Some possess a 3′–5′ exonucleolytic proofreading activity.

Strand displacement synthesis

An activity possessed by some DNA polymerases that involves displacement of the downstream DNA to produce a flap.

Proliferating cell nuclear antigen

(PCNA). A homotrimeric sliding clamp complex that plays an essential part at the replication fork through recruitment of many enzymes required for DNA replication and repair. Post-translational modifications of PCNA initiate specific DNA repair processes that are crucial for maintaining genome stability.

Replication stress

A cascade of responses that result from difficulties during DNA synthesis, such as polymerase stalling at a DNA lesion in the template strand. Replication stress may slow replisome progression and sensitizes cells to exogenous replication stress-inducing agents.

Micronuclei

Aberrant nuclear structures located within the cytoplasm that are composed of chromosomal fragments that were not properly incorporated into a daughter nucleus during cell division. Micronuclei are indicative of DNA damage.

γH2AX foci

Nuclear foci that serve as biomarkers for DNA double-strand breaks (DSBs), formed by a variant of histone H2A that is phosphorylated at Ser139 by checkpoint kinases during DNA damage response activation and modified over a large region of chromatin in the vicinity of a DSB.

CMG complex

(Cdc45/Mcm2-7/GINS complex). The helicase that unwinds DNA at eukaryotic replication forks. It is physically associated with the leading strand DNA polymerase Pol ε.

Post-replication DNA repair

A process that occurs at sites of DNA damage to facilitate the completion of replication using error-free (MMS2-dependent template switching) or error-prone (DNA polymerase ζ-dependent translesion synthesis) DNA damage-tolerance pathways.

RNA–DNA damage

Genome instability caused by the presence of ribonucleotides in DNA. RNA–DNA damage is associated with mutagenesis, chromosomal rearrangements, replication stress and DNA breaks.

Gene conversion events

DNA repair events that involve the transfer of genetic information from a donor sequence to a homologous recipient, such that the two become identical.

Loss of heterozygosity

(LOH). A chromosomal rearrangement in diploid cells that occurs when the template for DNA synthesis during homologous recombination repair is a homologous chromosome. This may result in loss of a functional allele and occurs frequently in tumour cells.

Mitotic interhomologue recombination

Mitotic crossover recombination that occurs between homologous chromosomes during the repair of a DNA lesion.

Non-allelic homologous recombination

(NAHR). A form of homologous recombination that occurs between similar sequences, causing chromosomal translocations and copy number variation.

DNA fragile site

A specific genomic locus that is susceptible to spontaneous DNA breaks, which may result in chromosomal rearrangements and contribute to human disease.

G4 quadruplex

A higher-order DNA structural motif composed of four strands of guanine-rich sequences that form a stable planar stacked structure at specific genomic locations, including telomeres and gene promoters, where they seem to have important positive and negative biological roles.

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Williams, J., Lujan, S. & Kunkel, T. Processing ribonucleotides incorporated during eukaryotic DNA replication. Nat Rev Mol Cell Biol 17, 350–363 (2016). https://doi.org/10.1038/nrm.2016.37

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