rNMPs In DNA Research Articles

RNMPID: a database for riboNucleoside MonoPhosphates in DNA.

Ribonucleoside monophosphates (rNMPs) are the most abundant non-standard nucleotides embedded in genomic DNA. If the presence of rNMP in DNA cannot be controlled, it can lead to genome instability. The actual regulatory functions of rNMPs in DNA remain mainly unknown. Considering the association between rNMP embedment and various diseases and cancer, the phenomenon of rNMP embedment in DNA has become a prominent area of research in recent years. We introduce the rNMPID database, which is the first database revealing rNMP-embedment characteristics, strand bias, and preferred incorporation patterns in the genomic DNA of samples from bacterial to human cells of different genetic backgrounds. The rNMPID database uses datasets generated by different rNMP-mapping techniques. It provides the researchers with a solid foundation to explore the features of rNMP embedded in the genomic DNA of multiple sources, and their association with cellular functions, and, in future, disease. It also significantly benefits researchers in the fields of genetics and genomics who aim to integrate their studies with the rNMP-embedment data. rNMPID is freely accessible on the web at https://www.rnmpid.org.

Processing of matched and mismatched rNMPs in DNA by archaeal ribonucleotide excision repair

Processing of matched and mismatched rNMPs in DNA by archaeal ribonucleotide excision repair

Disproportionate presence of adenosine in mitochondrial and chloroplast DNA of Chlamydomonas reinhardtii

SummaryRibonucleoside monophosphates (rNMPs) represent the most common non-standard nucleotides found in the genome of cells. The distribution of rNMPs in DNA has been studied only in limited genomes. Using the ribose-seq protocol and the Ribose-Map bioinformatics toolkit, we reveal the distribution of rNMPs incorporated into the whole genome of a photosynthetic unicellular green alga, Chlamydomonas reinhardtii. We discovered a disproportionate incorporation of adenosine in the mitochondrial and chloroplast DNA, in contrast to the nuclear DNA, relative to the corresponding nucleotide content of these C. reinhardtii organelle genomes. Our results demonstrate that the rNMP content in the DNA of the algal organelles reflects an elevated ATP level present in the algal cells. We reveal specific biases and patterns in rNMP distributions in the algal mitochondrial, chloroplast, and nuclear DNA. Moreover, we identified the C. reinhardtii orthologous genes for all three subunits of the RNase H2 enzyme using GeneMark-EP + gene finder.

Unlike the Escherichia coli counterpart, archaeal RNase HII cannot process ribose monophosphate abasic sites and oxidized ribonucleotides embedded in DNA

The presence of ribonucleoside monophosphates (rNMPs) in nuclear DNA decreases genome stability. To ensure survival despite rNMP insertions, cells have evolved a complex network of DNA repair mechanisms, in which the ribonucleotide excision repair pathway, initiated by type 2 RNase H (RNase HII/2), plays a major role. We recently demonstrated that eukaryotic RNase H2 cannot repair damage, that is, ribose monophosphate abasic (both apurinic or apyrimidinic) site (rAP) or oxidized rNMP embedded in DNA. Currently, it remains unclear why RNase H2 is unable to repair these modified nucleic acids having either only a sugar moiety or an oxidized base. Here, we compared the endoribonuclease specificity of the RNase HII enzymes from the archaeon Pyrococcus abyssi and the bacterium Escherichia coli, examining their ability to process damaged rNMPs embedded in DNA in vitro We found that E. coli RNase HII cleaves both rAP and oxidized rNMP sites. In contrast, like the eukaryotic RNase H2, P. abyssi RNase HII did not display any rAP or oxidized rNMP incision activities, even though it recognized them. Notably, the archaeal enzyme was also inactive on a mismatched rNMP, whereas the E. coli enzyme displayed a strong preference for the mispaired rNMP over the paired rNMP in DNA. On the basis of our biochemical findings and also structural modeling analyses of RNase HII/2 proteins from organisms belonging to all three domains of life, we propose that RNases HII/2's dual roles in ribonucleotide excision repair and RNA/DNA hydrolysis result in limited acceptance of modified rNMPs embedded in DNA.

Two RNase H2 Mutants with Differential rNMP Processing Activity Reveal a Threshold of Ribonucleotide Tolerance for Embryonic Development

SUMMARYRNase H2 has two distinct functions: initiation of the ribonucleotide excision repair (RER) pathway by cleaving ribonucleotides (rNMPs) incorporated during DNA replication and processing the RNA portion of an R-loop formed during transcription. An RNase H2 mutant lacking RER activity but supporting R-loop removal revealed that rNMPs in DNA initiate p53-dependent DNA damage response and early embryonic arrest in mouse. However, an RNase H2 AGS-related mutant with residual RER activity develops to birth. Estimations of the number of rNMPs in DNA in these two mutants define a ribonucleotide threshold above which p53 induces apoptosis. Below the threshold, rNMPs in DNA trigger an innate immune response. Compound heterozygous cells, containing both defective enzymes, retain rNMPs above the threshold, indicative of competition for RER substrates between active and inactive enzymes, suggesting that patients with compound heterozygous mutations in RNASEH2 genes may not reflect the properties of recombinantly expressed proteins.

Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2.

Ribonucleoside 5′-monophosphates (rNMPs) are the most common non-standard nucleotides found in DNA of eukaryotic cells, with over 100 million rNMPs transiently incorporated in the mammalian genome per cell cycle. Human ribonuclease (RNase) H2 is the principal enzyme able to cleave rNMPs in DNA. Whether RNase H2 may process abasic or oxidized rNMPs incorporated in DNA is unknown. The base excision repair (BER) pathway is mainly responsible for repairing oxidized and abasic sites into DNA. Here we show that human RNase H2 is unable to process an abasic rNMP (rAP site) or a ribose 8oxoG (r8oxoG) site embedded in DNA. On the contrary, we found that recombinant purified human apurinic/apyrimidinic endonuclease-1 (APE1) and APE1 from human cell extracts efficiently process an rAP site in DNA and have weak endoribonuclease and 3′-exonuclease activities on r8oxoG substrate. Using biochemical assays, our results provide evidence of a human enzyme able to recognize and process abasic and oxidized ribonucleotides embedded in DNA.

Increased Spontaneous Recombination in RNase H2-Deficient Cells Arises From Multiple Contiguous rNMPs and Not From Single rNMP Residues Incorporated by DNA Polymerase Epsilon.

Ribonucleotides can become embedded in DNA from insertion by DNA polymerases, failure to remove Okazaki fragment primers, R-loops that can prime replication, and RNA/cDNA-mediated recombination. RNA:DNA hybrids are removed by RNase H enzymes. Single rNMPs in DNA are removed by RNase H2 and if they remain on the leading strand, can lead to mutagenesis in a Top1-dependent pathway. rNMPs in DNA can also stimulate genome instability, among which are homologous recombination gene conversion events. We previously found that, similar to the rNMP-stimulated mutagenesis, rNMP-stimulated recombination was also Top1-dependent. However, in contrast to mutagenesis, we report here that recombination is not stimulated by rNMPs incorporated by the replicative polymerase epsilon. Instead, recombination seems to be stimulated by multiple contiguous rNMPs, which may arise from R-loops or replication priming events.

RNase H2 roles in genome integrity revealed by unlinking its activities.

Ribonuclease H2 (RNase H2) protects genome integrity by its dual roles of resolving transcription-related R-loops and ribonucleotides incorporated in DNA during replication. To unlink these two functions, we generated a Saccharomyces cerevisiae RNase H2 mutant that can resolve R-loops but cannot cleave single ribonucleotides in DNA. This mutant definitively correlates the 2–5 bp deletions observed in rnh201Δ strains with single rNMPs in DNA. It also establishes a connection between R-loops and Sgs1-mediated replication reinitiation at stalled forks and identifies R-loops uniquely processed by RNase H2. In mouse, deletion of any of the genes coding for RNase H2 results in embryonic lethality, and in humans, RNase H2 hypomorphic mutations cause Aicardi–Goutières syndrome (AGS), a neuroinflammatory disorder. To determine the contribution of R-loops and rNMP in DNA to the defects observed in AGS, we characterized in yeast an AGS-related mutation, which is impaired in processing both substrates, but has sufficient R-loop degradation activity to complement the defects of rnh201Δ sgs1Δ strains. However, this AGS-related mutation accumulates 2–5 bp deletions at a very similar rate as the deletion strain.

Mispaired rNMPs in DNA are mutagenic and are targets of mismatch repair and RNases H

Numerous studies have shown that ribonucleoside monophosphates (rNMPs) are probably abundant among all nonstandard nucleotides occurring in genomic DNA. Therefore, it is important to understand to what extent rNMPs may alter genome integrity and what factors affect their stability. We developed oligonucleotide-driven gene correction assays in Escherichia coli and Saccharomyces cerevisiae to show that mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis and produce a genetic change. We discovered that isolated mispaired rNMPs in chromosomal DNA are removed by the mismatch repair system in competition with RNase H type 2. However, a mismatch within an RNA-DNA heteroduplex region requires RNase H type 1 for removal. In the absence of mismatch repair and RNases H, ribonucleotide-driven gene modification increased by a factor of 47 in yeast and 77,000 in E. coli.

The Major Roles of DNA Polymerases Epsilon and Delta at the Eukaryotic Replication Fork Are Evolutionarily Conserved

Coordinated replication of eukaryotic genomes is intrinsically asymmetric, with continuous leading strand synthesis preceding discontinuous lagging strand synthesis. Here we provide two types of evidence indicating that, in fission yeast, these two biosynthetic tasks are performed by two different replicases. First, in Schizosaccharomyces pombe strains encoding a polδ-L591M mutator allele, base substitutions in reporter genes placed in opposite orientations relative to a well-characterized replication origin are strand-specific and distributed in patterns implying that Polδ is primarily involved in lagging strand replication. Second, in strains encoding a polε-M630F allele and lacking the ability to repair rNMPs in DNA due to a defect in RNase H2, rNMPs are selectively observed in nascent leading strand DNA. The latter observation demonstrates that abundant rNMP incorporation during replication can be tolerated and that they are normally removed in an RNase H2-dependent manner. This provides strong physical evidence that Polε is the primary leading strand replicase. Collectively, these data and earlier results in budding yeast indicate that the major roles of Polδ and Polε at the eukaryotic replication fork are evolutionarily conserved.

Mismatch repair-independent tandem repeat sequence instability resulting from ribonucleotide incorporation by DNA polymerase ɛ

During DNA synthesis in vitro using dNTP and rNTP concentrations present in vivo, yeast replicative DNA polymerases α, δ and ɛ (Pols α, δ and ɛ) stably incorporate rNTPs into DNA. rNTPs are also incorporated during replication in vivo, and they are repaired in an RNase H2-dependent manner. In strains encoding a mutator allele of Pol ɛ ( pol2-M644G), failure to remove rNMPs from DNA due to deletion of the RNH201 gene encoding the catalytic subunit of RNase H2, results in deletion of 2–5 base pairs in short repetitive sequences. Deletion rates depend on the orientation of the reporter gene relative to a nearby replication origin, suggesting that mutations result from rNMPs incorporated during replication. Here we demonstrate that 2–5 base pair deletion mutagenesis also strongly increases in rnh201Δ strains encoding wild type DNA polymerases. As in the pol2-M644G strains, the deletions occur at repetitive sequences and are orientation-dependent, suggesting that mismatches involving misaligned strands arise that could be subject to mismatch repair. Unexpectedly however, 2–5 base pair deletion rates resulting from loss of RNH201 in the pol2-M644G strain are unaffected by concomitant loss of MSH3, MSH6, or both. It could be that the mismatch repair machinery is unable to repair mismatches resulting from unrepaired rNMPs incorporated into DNA by M644G Pol ɛ, but this possibility is belied by the observation that Msh2–Msh6 can bind to a ribonucleotide-containing mismatch. Alternatively, following incorporation of rNMPs by M644G Pol ɛ during replication, the conversion of unrepaired rNMPs into mutations may occur outside the context of replication, e.g., during the repair of nicks resulting from rNMPs in DNA. The results make interesting predictions that can be tested.

Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases

Measurements of nucleoside triphosphate levels in Saccharomyces cerevisiae reveal that the four rNTPs are in 36- to 190-fold molar excess over their corresponding dNTPs. During DNA synthesis in vitro using the physiological nucleoside triphosphate concentrations, yeast DNA polymerase epsilon, which is implicated in leading strand replication, incorporates one rNMP for every 1,250 dNMPs. Pol delta and Pol alpha, which conduct lagging strand replication, incorporate one rNMP for every 5,000 or 625 dNMPs, respectively. Discrimination against rNMP incorporation varies widely, in some cases by more than 100-fold, depending on the identity of the base and the template sequence context in which it is located. Given estimates of the amount of replication catalyzed by Pols alpha, delta, and epsilon, the results are consistent with the possibility that more than 10,000 rNMPs may be incorporated into the nuclear genome during each round of replication in yeast. Thus, rNMPs may be the most common noncanonical nucleotides introduced into the eukaryotic genome. Potential beneficial and negative consequences of abundant ribonucleotide incorporation into DNA are discussed, including the possibility that unrepaired rNMPs in DNA could be problematic because yeast DNA polymerase epsilon has difficulty bypassing a single rNMP present within a DNA template.