In recent years it has become clear that RNases H are major players in the maintenance of genome stability in all organisms. Their roles in processing two types of nucleic acid structures that unchecked can induce replicative stress and DNA damage made these enzymes a focus of research in a growing number of labs. RNA/DNA hybrids and their more common form in cells, R-loops, are substrates for both RNase H1 and RNase H2 and when not removed because RNases H are defective can hinder the progression of the replication fork and induce single and double strand DNA breaks. Single ribonucleotides incorporated in genomic DNA by replicative polymerases are replaced by deoxyribonucleotides in a process initiated by RNase H2, and if left in DNA can cause DNA damage in a Topoisomerase-dependent manner. Mammalian embryos cannot progress to birth in the absence of RNase H1 or RNase H2; both enzymes are essential during embryogenesis. However, yeasts and prokaryotes such as Escherichia coli can survive well without RNase H1 or RNase H2, and in the case of yeast even without both enzymes, while in E. coli the absence of RNase HI and HII is temperature sensitive for growth. Thus, yeast and E. coli have been widely used to study the functions of these enzymes and in this issue several authors report on their works using these systems to elucidate the effect of mutations in RNase H1 and/or H2. Although the role of RNase H1 in processing R-loops in the nucleus is well documented, the embryonic lethality in mouse lacking RNase H1 is due to mitochondrial DNA replication defects, and in this issue the article by Ian Holt explains the mechanism of mitochondrial DNA replication and the role of RNase H1 in RNA primer processing. A good understanding of any enzymatic function requires a clear knowledge of the enzyme’s structure and in the review of Hyjek, Figiel, and Nowotny we get to learn the mechanisms of substrate recognition and cleavage by the different classes of RNases H. Finally, the interplay between RNases H and other enzymes that process RNA/DNA hybrids, such as helicases are explored in the article by Pohl and Zakian. In the following text, we provide a brief synopsis of the different studies that comprise this Special Issue. This synopsis follows the order in which the articles appear in the Special Issue.
Marc Drolet and Julie Brochu reviewed the role of R-loops in DNA replication in the absence of RNase HI in E. coli. While initiation of DNA replication is tightly regulated by DnaA to occur only at oriC, when rnhA is mutated DNA synthesis can start in the absence of DnaA/oriC at any region where there is an R-loop by using the RNA strand as primer for synthesis by DNA Pol I. They suggest that in E. coli, the regulation of replication from R-loops is an important function of RNase HI and also of type 1A topoisomerases, which control DNA supercoiling that favors R-loops formation. Unregulated replication appears to be a major mechanism by which R-loops threaten the stability of the genome in E. coli, although they might be also beneficial by promoting stress-induced mutagenesis that can contribute to bacterial adaptation to stress.
In their manuscript entitled “Role of RNase H enzymes in maintaining genome stability in Escherichia coli expressing a steric-gate mutant of pol VICE391” Walsh et al., elucidated pathways of ribonucleotide excision repair (RER) in E. coli by utilizing a steric gate variant of pol VICE391. Similar to E. coli DNA polymerase V, pol VICE391 is a heterotrimeric complex comprised of RumA′2B proteins, but it has a higher “SOS mutator” polymerase activity than E. coli pol V due in part to the enhanced longevity of pol VICE391 in replication foci as well as to a greater processivity. Using strains with defects in RER and containing steric gate variant of pol VICE391 and pol V, the authors confirm that RNase HII, primarily operating on the leading strand, catalyzes the major RER pathway, while Nucleotide Excision Repair (NER) and RNase HI-dependent repair play backup roles that are mainly confined to the lagging strand.
Williams et al. performed genome-wide analysis of mutation rates and specificity in RNase H2-deficient yeast strains expressing mutant alleles of the DNA polymerases that confer increased ribonucleotide incorporation into DNA. Their analysis established that ribonucleotide-dependent deletion mutagenesis occurs across the yeast genome and is TOP1-dependent. More specifically, they determined that AG and CA dinucleotide deletions are preferentially elevated, and di- and trinucleotide repeat deletion rates increase exponentially with tract length.
Potenski et al. modeled some of the human RNase H2 mutations found in Aicardi-Goutières syndrome (AGS) in the yeast RNase H2 enzyme. They found that not all human AGS alleles have phenotypes related to genome instability in yeast. This may be because some human AGS mutations are seen only as compound heterozygotes and the other allele may have the stronger phenotype. However, some human AGS alleles have a very strong phenotype in yeast, and those would be candidates for studies of genome instability in AGS cells. They found that the in vivo genome instability phenotypes of the AGS mutants modeled in yeast correlated with increased retention of riNbonucleotides in DNA.
The Pohl and Zakian article reviews the many functions of Pif1, Rrm3 and Pfh1 helicases and highlights those that might be involved in R-loop removal, such as during replication of highly transcribed RNA POL II and III genes, centromeres, and inhibition of telomerase. Although R-loops are probably resolved primarily by RNase H enzymes, DNA helicases in combination with RNA nucleases, can also dismantle and degrade R-loops. Pif1 family helicases are virtually ubiquitous in eukaryotes and have 5′ to 3′ DNA helicase activity that is particularly proficient at displacing RNA from an RNA/DNA hybrid and in unwinding G-quadruplex DNA, structures that are often associated with R-loop formation. The two budding yeast Pif1 family helicases, Pif1 and Rrm3, and the single fission yeast family member, Pfh1, are multiple functional helicases that promote genome stability of both nuclear and mitochondrial DNA.
Ian Holt reviews the roles of RNase H1 in mammals. RNase H1 is critical for mitochondrial DNA replication, as it has an essential role in removing RNA primers. RNase H1 also likely acts at the advancing replication fork and may be a component of the mitochondrial replisome. This would enable the enzyme to facilitate the proposed transcript-dependent mechanism of mitochondrial DNA replication. Pathological mutant forms of RNase H1 have been identified that cause mitochondrial diseases associated with multiple deletions of mitochondrial DNA, similar to mutant forms of the mitochondrial DNA polymerase and helicase. Pathological mutant variants of RNase H1 slow or stall mtDNA replication, which could explain the formation of deleted mitochondrial DNA molecules, as could a position in the replisome. However, there is little or no evidence that the disease stems from unprocessed primers. Another potential contribution to the disease mechanism is the inappropriate degradation of legitimate RNA/DNA hybrids, in particular a major species the mitochondrial R-loop that spans much of the major non-coding region and which is expected to have multiple roles in mitochondrial DNA replication and maintenance.
Malwina Hyjek, Małgorzata Figiel and Marcin Nowotny review summarizes the structural basis of substrate recognition and enzymatic cleavage by different classes of RNases H. Biochemical and structural studies of these enzymes led to comprehensive understanding of their mechanism of action and provided insights into general principles of nucleic acid recognition and hydrolysis. To achieve specificity toward RNA and DNA the three types of RNases H exploit different dynamic properties (flexibility) of the two types of nucleic acids. In addition, specific substrate binding is mediated by electrostatic interactions between the protein and 2′–OH groups of RNA and DNA-specific stacking interactions with deoxyribose rings.
Beáta Boros-Oláh et al. described a potential link between RNA/DNA hybrids and R-loops accumulation and tumorigenesis. They suggest that R-loops and R-loop binding proteins may be relevant epigenetic markers and therapeutic targets in multiple cancer types. It has been recognized that uncontrolled R-loops are a hazard to genome integrity, and in this paper, they corelate the expression of RNA/DNA binding proteins in various cancer types with survival and therapeutic responses.
Susana Cerritelli and Robert Crouch review the contribution of the RNaseH2-RED mutant to the understanding of the RNase H2 functions in eukaryotes. This mutant can process RNA/DNA hybrids but is unable to cleave ribonucleotides embedded in DNA, so its name RNase H2-RED (Ribonucleotide Excision Defective). Multiple studies in yeast using this mutant enzyme have revealed that both activities of RNase H2 are necessary to maintain genome integrity, although depending on the strain background and the abundance of the substrates one activity can take precedent over the other. In mouse RNase H2-RED confirmed that ribonucleotides in DNA induce lethality during embryogenesis in a p53-dependent DNA damage response.