Depletion of RNASEH2 Activity Leads to Accumulation of DNA Double-strand Breaks and Reduced Cellular Survivability in T Cell Leukemia.

Ribonuclease H2 (RNase H2) is a member of the ribonuclease H family of enzymes involved in removal of RNA from RNA-DNA hybrids as well as ribonucleotides which get misincorporated into the genomic DNA. Recent studies have shown that RNase H2 function is also needed for successful DNA repair through NHEJ events where DNA pol µ uses ribonucleotides during the gap filling stage. Mammalian RNase H2 is composed of three subunits, RNASEH2A, RNASEH2B and RNASEH2C. There have been studies suggesting changes in expression of these genes in various cancers of breast, prostate, colon, liver, and kidney. In this study, we have investigated the functional role of RNASEH2A and RNASEH2B in leukemic T-cells, MOLT4 and Jurkat. shRNA mediated knockdown of RNASEH2A/ RNASEH2B expression led to reduced cell survival and increase in apoptotic cell population. Importantly, knockdown of RNASEH2A or RNASEH2B, led to cell cycle arrest at S phase and increased number of 53BP1 foci due to abrogation of NHEJ. Interestingly, RNASEH2A or RNASEH2B depleted cells showed significantly retarded DSB repair kinetics compared to scrambled shRNA control, when exposed to ionizing radiation suggesting that NHEJ is abrogated due to loss of RNASEH2 activity in T-ALL cells. Thus, we uncover the importance of RNase H2 function in leukemic cells and suggest that it can be targeted for cancer therapy.

TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution.

R-loops are three-stranded, RNA-DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.

RNA-cDNA hybrids mediate transposition via different mechanisms.

Retrotransposons can represent half of eukaryotic genomes. Retrotransposon dysregulation destabilizes genomes and has been linked to various human diseases. Emerging regulators of retromobility include RNA-DNA hybrid-containing structures known as R-loops. Accumulation of these structures at the transposons of yeast 1 (Ty1) elements has been shown to increase Ty1 retromobility through an unknown mechanism. Here, via a targeted genetic screen, we identified the rnh1Δ rad27Δ yeast mutant, which lacked both the Ty1 inhibitor Rad27 and the RNA-DNA hybrid suppressor Rnh1. The mutant exhibited elevated levels of Ty1 cDNA-associated RNA-DNA hybrids that promoted Ty1 mobility. Moreover, in this rnh1Δ rad27Δ mutant, but not in the double RNase H mutant rnh1Δ rnh201Δ, RNA-DNA hybrids preferentially existed as duplex nucleic acid structures and increased Ty1 mobility in a Rad52-dependent manner. The data indicate that in cells lacking RNA-DNA hybrid and Ty1 repressors, elevated levels of RNA-cDNA hybrids, which are associated with duplex nucleic acid structures, boost Ty1 mobility via a Rad52-dependent mechanism. In contrast, in cells lacking RNA-DNA hybrid repressors alone, elevated levels of RNA-cDNA hybrids, which are associated with triplex nucleic acid structures, boost Ty1 mobility via a Rad52-independent process. We propose that duplex and triplex RNA-DNA hybrids promote transposon mobility via Rad52-dependent or -independent mechanisms.

Development of a ribonuclease containing a G4-specific binding motif for programmable RNA cleavage.

We developed a ribonuclease for site-specific targeting and cleavage of single-stranded RNA. The engineered RNase protein was constructed by incorporating two independent functional domains, an RNase HI domain that could cleave the RNA strand in a DNA-RNA hybrid, and a domain of the RHAU protein that could selectively recognize a parallel DNA G-quadruplex (G4). The newly designed RNase first recruits a DNA guide oligonucleotide containing both a parallel G4 motif and a template sequence complementary to the target RNA. This RNase:DNA complex targets and efficiently cleaves the single-stranded RNA in a site-specific manner. A major cleavage site occurs at the RNA region that is complementary to the DNA template sequence. The newly designed RNase can serve as a simple tool for RNA manipulation and probing RNA structure.

R-Loop Depletion by Over-expressed RNase H1 in Mouse B Cells Increases Activation-Induced Deaminase Access to the Transcribed Strand without Altering Frequency of Isotype Switching.

R-loops, three-strand structures consisting of mRNA hybridized to the complementary DNA and a single-stranded DNA loop, are formed in switch regions on the heavy-chain immunoglobulin locus. To determine if R-loops have a direct effect on any of the steps involved in isotype switching, we generated a transgenic mouse that over-expressed RNase H1, an enzyme that cleaves the RNA of RNA/DNA hybrids in B cells. R-loops in the switch µ region were depleted by 70% in ex vivo activated splenic B cells. Frequencies of isotype switching to IgG1, IgG2b, IgG2c, and IgG3 were the same as C57BL/6 control cells. However, somatic hypermutation was increased specifically on the transcribed strand from µ-γ joins, indicating that R-loops limit activation-induced (cytosine) deaminase access to the transcribed DNA strand. Our data suggest that, in the normal G+C-rich context of mammalian class switch recombination regions, R-loops are obligatory intermediates. Processing of the R-loops is needed to remove RNA allowing activation-induced (cytosine) deaminase to promote somatic hypermutation on both DNA strands to generate double-strand DNA breaks for efficient class switch recombination. One of the two cellular RNases H may assist in this process.

Allele-Selective Inhibition of Mutant Huntingtin with 2-Thio- and C5- Triazolylphenyl-Deoxythymidine-Modified Antisense Oligonucleotides.

We report the effect of introducing a single incorporation of 2-thio-deoxythymidine (2S-dT) or C5-Triazolylphenyl-deoxythymidine (5-TrPh-dT) at four positions within the gap region of RNase H gapmer antisense oligonucleotides (ASOs) for reducing wild-type and mutant huntingtin mRNA in human patient fibroblasts. We show that these modifications can modulate processing of the ASO/RNA heteroduplexes by recombinant human RNase H1 in a position-dependent manner. We also created a structural model of the catalytic domain of human RNase H bound to ASO/RNA heteroduplexes to rationalize the activity and selectivity observations in cells and in the biochemical assays. Our results highlight the ability of chemical modifications in the gap region to produce profound changes in ASO behavior.

Structural and inhibition studies of the RNase H function of xenotropic murine leukemia virus-related virus reverse transcriptase.

RNase H inhibitors (RNHIs) have gained attention as potential HIV-1 therapeutics. Although several RNHIs have been studied in the context of HIV-1 reverse transcriptase (RT) RNase H, there is no information on inhibitors that might affect the RNase H activity of other RTs. We performed biochemical, virological, crystallographic, and molecular modeling studies to compare the RNase H function and inhibition profiles of the gammaretroviral xenotropic murine leukemia virus-related virus (XMRV) and Moloney murine leukemia virus (MoMLV) RTs to those of HIV-1 RT. The RNase H activity of XMRV RT is significantly lower than that of HIV-1 RT and comparable to that of MoMLV RT. XMRV and MoMLV, but not HIV-1 RT, had optimal RNase H activities in the presence of Mn²âº and not Mg²âº. Using hydroxyl-radical footprinting assays, we demonstrated that the distance between the polymerase and RNase H domains in the MoMLV and XMRV RTs is longer than that in the HIV-1 RT by ∼3.4 Å. We identified one naphthyridinone and one hydroxyisoquinolinedione as potent inhibitors of HIV-1 and XMRV RT RNases H with 50% inhibitory concentrations ranging from ∼0.8 to 0.02 µM. Two acylhydrazones effective against HIV-1 RT RNase H were less potent against the XMRV enzyme. We also solved the crystal structure of an XMRV RNase H fragment at high resolution (1.5 Å) and determined the molecular details of the XMRV RNase H active site, thus providing a framework that would be useful for the design of antivirals that target RNase H.

RNase H and postreplication repair protect cells from ribonucleotides incorporated in DNA.

The chemical identity and integrity of the genome is challenged by the incorporation of ribonucleoside triphosphates (rNTPs) in place of deoxyribonucleoside triphosphates (dNTPs) during replication. Misincorporation is limited by the selectivity of DNA replicases. We show that accumulation of ribonucleoside monophosphates (rNMPs) in the genome causes replication stress and has toxic consequences, particularly in the absence of RNase H1 and RNase H2, which remove rNMPs. We demonstrate that postreplication repair (PRR) pathways-MMS2-dependent template switch and Pol ζ-dependent bypass-are crucial for tolerating the presence of rNMPs in the chromosomes; indeed, we show that Pol ζ efficiently replicates over 1-4 rNMPs. Moreover, cells lacking RNase H accumulate mono- and polyubiquitylated PCNA and have a constitutively activated PRR. Our findings describe a crucial function for RNase H1, RNase H2, template switch, and translesion DNA synthesis in overcoming rNTPs misincorporated during DNA replication, and may be relevant for the pathogenesis of Aicardi-Goutières syndrome.

[Positive and negative regulation of transcription from HIV provirus].

The RNA genome of retroviruses including human immunodeficiency virus type 1 (HIV-1) will be converted into DNA, called "propvirus". This proviral DNA will be integrated into host cell genome and behave like host genes. Since the step at which the viral RNA genome is converted into DNA will not allow any increase of viral genetic information because of the presence of RNaseH activity inherent to the reverse transcriptase and is responsible for the degradation of viral RNA in forming the DNA:RNA hybrid as the intermediate molecule for this conversion. However, during transcription from proviral DNA into viral RNA, hundreds and even thousands of mRNA encoding viral information will be synthesized by the action of host cellular RNA polymerase II, thus producing a large amount of progeny viral particles after translation and assembly. HIV is unique in that it contains virus-specific transcriptional activator called Tat.

Retron Se72 utilizes a unique strategy of the self-priming initiation of reverse transcription.

Unlike all of the other retrons, the bacterial retron reverse transcriptase RrtE is capable of synthesizing small double-stranded DNA (sdsDNA) from template RNA. In this study, we analyzed the biosynthesis of the sdsDNA by RrtE in detail. We found out that the initiation of reverse transcription was dependent on a novel self-priming mechanism utilizing a free 3'OH of RNA that is reverse-transcribed into sdsDNA. The priming of the sdsDNA synthesis was not dependent on any particular nucleotide being used as a donor of 3'OH (unlike all of the other retrons, which prime from 2'OH of a particular guanosine) or any particular nucleotide being introduced into the sdsDNA first. Due to the relaxed demands for the initiation of reverse transcription, RrtE has the potential to generate dsDNA from different RNA transcripts in vivo.

Synthetic in vitro transcriptional oscillators.

The construction of synthetic biochemical circuits from simple components illuminates how complex behaviors can arise in chemistry and builds a foundation for future biological technologies. A simplified analog of genetic regulatory networks, in vitro transcriptional circuits, provides a modular platform for the systematic construction of arbitrary circuits and requires only two essential enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H, to produce and degrade RNA signals. In this study, we design and experimentally demonstrate three transcriptional oscillators in vitro. First, a negative feedback oscillator comprising two switches, regulated by excitatory and inhibitory RNA signals, showed up to five complete cycles. To demonstrate modularity and to explore the design space further, a positive-feedback loop was added that modulates and extends the oscillatory regime. Finally, a three-switch ring oscillator was constructed and analyzed. Mathematical modeling guided the design process, identified experimental conditions likely to yield oscillations, and explained the system's robust response to interference by short degradation products. Synthetic transcriptional oscillators could prove valuable for systematic exploration of biochemical circuit design principles and for controlling nanoscale devices and orchestrating processes within artificial cells.

Retroviral integrase superfamily: the structural perspective.

The retroviral integrase superfamily (RISF) comprises numerous important nucleic acid-processing enzymes, including transposases, integrases and various nucleases. These enzymes are involved in a wide range of processes such as transposition, replication and repair of DNA, homologous recombination, and RNA-mediated gene silencing. Two out of the four enzymes that are encoded by the human immunodeficiency virus--RNase H1 and integrase--are members of this superfamily. RISF enzymes act on various substrates, and yet show remarkable mechanistic and structural similarities. All share a common fold of the catalytic core and the active site, which is composed primarily of carboxylate residues. Here, I present RISF proteins from a structural perspective, describing the individual members and the common and divergent elements of their structures, as well as the mechanistic insights gained from the structures of RNase H1 enzyme complexes with RNA/DNA hybrids.

Mechanism by which a glutamine to leucine substitution at residue 509 in the ribonuclease H domain of HIV-1 reverse transcriptase confers zidovudine resistance.

We recently reported that zidovudine (AZT) selected for the Q509L mutation in the ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT), which increases resistance to AZT in combination with the thymidine analogue mutations D67N, K70R, and T215F. In the current study, we have defined the mechanism by which Q509L confers AZT resistance by performing in-depth biochemical analyses of wild type, D67N/K70R/T215F and D67N/K70R/T215F/Q509L HIV-1 RT. Our results show that Q509L increases AZT-monophosphate (AZT-MP) excision activity of RT on RNA/DNA template/primers (T/Ps) but not DNA/DNA T/Ps. This increase in excision activity on the RNA/DNA T/P is due to Q509L decreasing a secondary RNase H cleavage event that reduces the RNA/DNA duplex length to 10 nucleotides and significantly impairs the enzyme's ability to excise the chain-terminating nucleotide. Presteady-state kinetic analyses indicate that Q509L does not affect initial rates of the polymerase-directed RNase H activity but only polymerase-independent cleavages that occur after a T/P dissociation event. Furthermore, competition binding assays suggest that Q509L decreases the affinity of the enzyme to bind T/P with duplex lengths less than 18 nucleotides in the polymerase-independent RNase H cleavage mode, while not affecting the enzyme's affinity to bind the same T/P in an AZT-MP excision competent mode. Taken together, this study provides the first mechanistic insights into how a mutation in the RNase H domain of RT increases AZT resistance and highlights how the polymerase and RNase H domains of RT function in concert to confer drug resistance.

Depletion of RNase HI activity in Escherichia coli lacking DNA topoisomerase I leads to defects in DNA supercoiling and segregation.

Gyrase-mediated hypernegative supercoiling is one manifestation of R-loop formation, a phenomenon that is normally suppressed by topoisomerase I (topA) in Escherichia coli. Overproduction of RNase HI (rnhA), an enzyme that removes the RNA moiety of R-loops, prevents hypernegative supercoiling and allows growth of topA null mutants. We previously showed that topA and rnhA null mutations are incompatible. We now report that such mutants were viable when RNase HI or topoisomerase III was expressed from a plasmid-borne gene. Surprisingly, DNA of topA null mutants became relaxed rather than hypernegatively supercoiled following depletion of RNase HI activity. This result failed to correlate with the cellular concentration of gyrase or topoisomerase IV (the other relaxing enzyme in the cell) or with transcription-induced supercoiling. Rather, intracellular DNA relaxation in the absence of RNase HI was related to inhibition of gyrase activity both in vivo and in extracts. Cells lacking topA and rnhA also exhibited properties consistent with segregation defects. Overproduction of topoisomerase III, an enzyme that can carry out DNA decatenation, corrected the segregation defects without restoring supercoiling activity. Collectively these data reveal (i) the existence of a cellular response to loss of RNase HI that counters the supercoiling activity of gyrase, and (ii) supercoiling-independent segregation defects due to loss of RNase HI from topA null mutants. Thus RNase HI plays a more central role in DNA topology than previously thought.

Structural, thermodynamic, and mutational analyses of a psychrotrophic RNase HI.

Ribonuclease (RNase) HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 was overproduced in Escherichia coli, purified, and structurally and biochemically characterized. The amino acid sequence of MR-1 RNase HI is 67% identical to that of E. coli RNase HI. The crystal structure of MR-1 RNase HI determined at 2.0 A resolution was highly similar to that of E. coli RNase HI, except that the number of intramolecular ion pairs and the fraction of polar surface area of MR-1 RNase HI were reduced compared to those of E. coli RNase HI. The enzymatic properties of MR-1 RNase HI were similar to those of E. coli RNase HI. However, MR-1 RNase HI was much less stable than E. coli RNase HI. The stability of MR-1 RNase HI against heat inactivation was lower than that of E. coli RNase HI by 19 degrees C. The conformational stability of MR-1 RNase HI was thermodynamically analyzed by monitoring the CD values at 220 nm. MR-1 RNase HI was less stable than E. coli RNase HI by 22.4 degrees C in Tm and 12.5 kJ/mol in DeltaG(H2O). The thermodynamic stability curve of MR-1 RNase HI was characterized by a downward shift and increased curvature, which results in an increased DeltaCp value, compared to that of E. coli RNase HI. Site-directed mutagenesis studies suggest that the difference in the number of intramolecular ion pairs partly accounts for the difference in stability between MR-1 and E. coli RNases HI.

Forced selection of a human immunodeficiency virus type 1 variant that uses a non-self tRNA primer for reverse transcription: involvement of viral RNA sequences and the reverse transcriptase enzyme.

Human immunodeficiency virus type 1 uses the tRNA(3)(Lys) molecule as a selective primer for reverse transcription. This primer specificity is imposed by sequence complementarity between the tRNA primer and two motifs in the viral RNA genome: the primer-binding site (PBS) and the primer activation signal (PAS). In addition, there may be specific interactions between the tRNA primer and viral proteins, such as the reverse transcriptase (RT) enzyme. We constructed viruses with mutations in the PAS and PBS that were designed to employ the nonself primer tRNA(Pro) or tRNA(1,2)(Lys). These mutants exhibited a severe replication defect, indicating that additional adaptation of the mutant virus is required to accommodate the new tRNA primer. Multiple independent virus evolution experiments were performed to select for fast-replicating variants. Reversion to the wild-type PBS-lys3 sequence was the most frequent escape route. However, we identified one culture in which the virus gained replication capacity without reversion of the PBS. This revertant virus eventually optimized the PAS motif for interaction with the nonself primer. Interestingly, earlier evolution samples revealed a single amino acid change of an otherwise well-conserved residue in the RNase H domain of the RT enzyme, implicating this domain in selective primer usage. We demonstrate that both the PAS and RT mutations improve the replication capacity of the tRNA(1,2)(Lys)-using virus.

Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs.

Although ribonuclease H activity has long been implicated as a molecular mechanism by which DNA-like oligonucleotides induce degradation of target RNAs, definitive proof that one or more RNase H is responsible is lacking. To date, two RNase H enzymes (H1 and H2) have been cloned and shown to be expressed in human cells and tissues. To determine the role of RNase H1 in the mechanism of action of DNA-like antisense drugs, we varied the levels of the enzyme in human cells and mouse liver and determined the correlation of those levels with the effects of a number of DNA-like antisense drugs. Our results demonstrate that in human cells RNase H1 is responsible for most of the activity of DNA-like antisense drugs. Further, we show that there are several additional previously undescribed RNases H in human cells that may participate in the effects of DNA-like antisense oligonucleotides.

The protein components and mechanism of eukaryotic Okazaki fragment maturation.

An initiator RNA (iRNA) is required to prime cellular DNA synthesis. The structure of double-stranded DNA allows the synthesis of one strand to be continuous but the other must be generated discontinuously. Frequent priming of the discontinuous strand results in the formation of many small segments, designated Okazaki fragments. These short pieces need to be processed and joined to form an intact DNA strand. Our knowledge of the mechanism of iRNA removal is still evolving. Early reconstituted systems suggesting that the removal of iRNA requires sequential action of RNase H and flap endonuclease 1 (FEN1) led to the RNase H/FEN1 model. However, genetic analyses implied that Dna2p, an essential helicase/nuclease, is required. Subsequent biochemical studies suggested sequential action of RPA, Dna2p, and FEN1 for iRNA removal, leading to the second model, the Dna2p/RPA/FEN1 model. Studies of strand-displacement synthesis by polymerase delta indicated that in a reconstituted system, FEN1 could act as soon as short flaps are created, giving rise to a third model, the FEN1-only model. Each of the three pathways is supported by different genetic and biochemical results. Properties of the major protein components in this process will be discussed, and the validity of each model as a true representation of Okazaki fragment processing will be critically evaluated in this review.

Antisense phosphorothioate oligodeoxyribonucleotide targeted against ICAM-1: synthetic and biological characterization of a process-related impurity formed during oligonucleotide synthesis.

A phosphorothioate-linked oligonucleotide bearing a 3'-terminal phosphorothioate monoester has been synthesized and characterized. This oligonucleotide has been identified as a process-related impurity formed during synthesis of ISIS 2302. Biological properties of the compound have been determined. Based on these data, it can be concluded that this species (3'-TPT) has biological properties similar to parent drug.

Failure to produce mitochondrial DNA results in embryonic lethality in Rnaseh1 null mice.

Although ribonucleases H (RNases H) have long been implicated in DNA metabolism, they are not required for viability in prokaryotes or unicellular eukaryotes. We generated Rnaseh1(-/-) mice to investigate the role of RNase H1 in mammals and observed developmental arrest at E8.5 in null embryos. A fraction of the mainly nuclear RNase H1 was targeted to mitochondria, and its absence in embryos resulted in a significant decrease in mitochondrial DNA content, leading to apoptotic cell death. This report links RNase H1 to generation of mitochondrial DNA, providing direct support for the strand-coupled mechanism of mitochondrial DNA replication. These findings also have important implications for therapy of mitochondrial dysfunctions and drug development for the structurally related RNase H of HIV.