RNA-DNA Junction Research Articles

Structures of LIG1 uncover the mechanism of sugar discrimination against 5′-RNA-DNA junctions during ribonucleotide excision repair

Ribonucleotides in DNA cause several types of genome instability and can be removed by ribonucleotide excision repair (RER) that is finalized by DNA ligase 1 (LIG1). However, the mechanism by which LIG1 discriminates the RER intermediate containing a 5'-RNA–DNA lesion generated by RNase H2-mediated cleavage of ribonucleotides remains unknown. Here, we determine X-ray structures of LIG1/5'-rG:C at the initial step of ligation where AMP is bound to the active site of the ligase, and uncover a large conformational change downstream the nick resulting in a shift at Arg(R)871 residue in the Adenylation domain of the ligase. Furthermore, we demonstrate a diminished ligation of the nick DNA substrate with a 5'-ribonucleotide in comparison to an efficient end joining of the nick substrate with a 3'-ribonucleotide by LIG1. Finally, our results demonstrate that mutations at the active site residues of the ligase and LIG1 disease-associated variants significantly impact the ligation efficiency of RNA–DNA heteroduplexes harboring “wrong” sugar at 3'- or 5'-end of nick. Collectively, our findings provide a novel atomic insight into proficient sugar discrimination by LIG1 during processing of the most abundant form of DNA damage in cells, genomic ribonucleotides, during initial step of the RER pathway.

A four-point molecular handover during Okazaki maturation.

DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the replicative DNA polymerase Pol IIIα terminates at a previously synthesized RNA primer, DNA Pol I takes over and continues DNA synthesis while displacing the downstream RNA primer. The displaced primer is subsequently excised by an endonuclease, followed by the sealing of the nick by a DNA ligase. Yet how the sequential actions of Pol IIIα, Pol I polymerase, Pol I endonuclease and DNA ligase are coordinated is poorly defined. Here we show that each enzymatic activity prepares the DNA substrate for the next activity, creating an efficient four-point molecular handover. The cryogenic-electron microscopy structure of Pol I bound to a DNA substrate with both an upstream and downstream primer reveals how it displaces the primer in a manner analogous to the monomeric helicases. Moreover, we find that in addition to its flap-directed nuclease activity, the endonuclease domain of Pol I also specifically cuts at the RNA-DNA junction, thus marking the end of the RNA primer and creating a 5' end that is a suitable substrate for the ligase activity of LigA once all RNA has been removed.

Large-Scale Photolithographic Synthesis of Chimeric DNA/RNA Hairpin Microarrays To Explore Sequence Specificity Landscapes of RNase HII Cleavage.

Ribonuclease HII (RNase HII) is an essential endoribonuclease that binds to double-stranded DNA with RNA nucleotide incorporations and cleaves 5′ of the ribonucleotide at RNA–DNA junctions. Thought to be present in all domains of life, RNase HII protects genomic integrity by initiating excision repair pathways that protect the encoded information from rapid degradation. There is sparse evidence that the enzyme cleaves some substrates better than others, but a large-scale study is missing. Such large-scale studies can be carried out on microarrays, and we employ chemical photolithography to synthesize very large combinatorial libraries of fluorescently labeled DNA/RNA chimeric sequences that self-anneal to form hairpin structures that are substrates for Escherichia coli RNase HII. The relative activity is determined by the loss of fluorescence upon cleavage. Each substrate includes a double-stranded 5 bp variable region with one to five consecutive ribonucleotide substitutions. We also examined the effect of all possible single and double mismatches, for a total of >9500 unique structures. Differences in cleavage efficiency indicate some level of substrate preference, and we identified the 5′-dC/rC-rA-dX-3′ motif in well-cleaved substrates. The results significantly extend known patterns of RNase HII sequence specificity and serve as a template using large-scale photolithographic synthesis to comprehensively map landscapes of substrate specificity of nucleic acid-processing enzymes.

A unique exonuclease ExoG cleaves between RNA and DNA in mitochondrial DNA replication.

Replication of sufficient mitochondrial DNA (mtDNA) is essential for maintaining mitochondrial functions in mammalian cells. During mtDNA replication, RNA primers must be removed before the nascent circular DNA strands rejoin. This process involves mitochondrial RNase H1, which removes most of the RNA primers but leaves two ribonucleotides attached to the 5′ end of nascent DNA. A subsequent 5′-exonuclease is required to remove the residual ribonucleotides, however, it remains unknown if any mitochondrial 5′-exonuclease could remove two RNA nucleotides from a hybrid duplex DNA. Here, we report that human mitochondrial Exonuclease G (ExoG) may participate in this particular process by efficiently cleaving at RNA–DNA junctions to remove the 5′-end RNA dinucleotide in an RNA/DNA hybrid duplex. Crystal structures of human ExoG bound respectively with DNA, RNA/DNA hybrid and RNA–DNA chimeric duplexes uncover the underlying structural mechanism of how ExoG specifically recognizes and cleaves at RNA–DNA junctions of a hybrid duplex with an A-form conformation. This study hence establishes the molecular basis of ExoG functioning as a unique 5′-exonuclease to mediate the flap-independent RNA primer removal process during mtDNA replication to maintain mitochondrial genome integrity.

Substrate Specificity for Bacterial RNases HII and HIII Is Influenced by Metal Availability.

We tested the activities of four predicated RNase H enzymes, including two RNase HI-type enzymes, in addition to RNase HII (RnhB) and RNase HIII (RnhC), on several RNA-DNA hybrid substrates with different divalent metal cations. We found that the two RNase HI-type enzymes, YpdQ and YpeP, failed to show activity on the three substrates tested. RNase HII and RNase HIII cleaved all the substrates tested, although the activity was dependent on the metal made available. We show that Bacillus subtilis RNase HII and RNase HIII are both able to incise 5' to a single ribonucleoside monophosphate (rNMP). We show that RNase HIII incision at a single rNMP occurs most efficiently with Mn2+, an activity we found to be conserved among other Gram-positive RNase HIII enzymes. Characterization of RNases HII and HIII with metal concentrations in the physiological range showed that RNase HII can cleave at single rNMPs embedded in DNA while RNase HIII is far less effective. Further, using metal concentrations within the physiological range, RNase HIII efficiently cleaved longer RNA-DNA hybrids lacking an RNA-DNA junction, while RNase HII was much less effective. Phenotypic analysis showed that cells with an rnhC deletion were sensitive to hydroxyurea (HU). In contrast, cells with an rnhB deletion showed wild-type growth in the presence of HU, supporting the hypothesis that RNases HII and HIII have distinct substrate specificities in vivo This work demonstrates how metal availability influences the substrate recognition and activity of RNases HII and HIII, providing insight into their functions in vivoIMPORTANCE RNase H represents a class of proteins that cleave RNA-DNA hybrids, helping resolve R-loops and Okazaki fragments, as well as initiating the process of ribonucleotide excision repair (RER). We investigated the activities of four Bacillus subtilis RNase H enzymes and found that only RNases HII and HIII have activity and that their substrate preference is dependent on metal availability. To understand the factors that contribute to RNase HII and RNase HIII substrate preference, we show that in the presence of metal concentrations within the physiological range, RNases HII and HIII have distinct activities on different RNA-DNA hybrids. This work provides insight into how RNases HII and HIII repair the broad range of RNA-DNA hybrids that form in Gram-positive bacteria.

RNase HII Saves rnhA Mutant Escherichia coli from R-Loop-Associated Chromosomal Fragmentation

The rnhAB mutant Escherichia coli, deficient in two RNase H enzymes that remove both R-loops and incorporated ribonucleotides (rNs) from DNA, grow slowly, suggesting accumulation of rN-containing DNA lesions (R-lesions). We report that the rnhAB mutants have reduced viability, form filaments with abnormal nucleoids, induce SOS, and fragment their chromosome, revealing replication and/or segregation stress. R-loops are known to interfere with replication forks, and sensitivity of the double rnhAB mutants to translation inhibition points to R-loops as precursors for R-lesions. However, the strict specificity of bacterial RNase HII for RNA–DNA junctions indicates that R-lesions have rNs integrated into DNA. Indeed, instead of relieving problems of rnhAB mutants, transient inhibition of replication from oriC kills them, suggesting that oriC-initiated replication removes R-loops instead of compounding them to R-lesions. Yet, replication from an R-loop-initiating plasmid origin kills the double rnhAB mutant, revealing generation of R-lesions by R-loop-primed DNA synthesis. These R-lesions could be R-tracts, contiguous runs of ≥4 RNA nucleotides within DNA strand and the only common substrate between the two bacterial RNase H enzymes. However, a plasmid relaxation test failed to detect R-tracts in DNA of the rnhAB mutants, although it readily detected R-patches (runs of 1–3 rNs). Instead, we detected R-gaps, single-strand gaps containing rNs, in the chromosomal DNA of the rnhAB mutant. Therefore, we propose that RNase H-deficient mutants convert some R-loops into R-tracts, which progress into R-gaps and then to double-strand breaks—explaining why R-tracts do not accumulate in RNase H-deficient cells, while double-strand breaks do.

An end-point method based on graphene oxide for RNase H analysis and inhibitors screening

As a highly conserved damage repair protein, RNase H can hydrolysis DNA-RNA heteroduplex endonucleolytically and cleave RNA-DNA junctions as well. In this study, we have developed an accurate and sensitive RNase H assay based on fluorophore-labeled chimeric substrate hydrolysis and the differential affinity of graphene oxide on RNA strand with different length. This end-point measurement method can detect RNase H in a range of 0.01 to 1 units /mL with a detection limit of 5.0×10-3 units/ mL under optimal conditions. We demonstrate the utility of the assay by screening antibiotics, resulting in the identification of gentamycin, streptomycin and kanamycin as inhibitors with IC50 of 60±5µM, 70±8µM and 300±20µM, respectively. Furthermore, the assay was reliably used to detect RNase H in complicated biosamples and found that RNase H activity in tumor cells was inhibited by gentamycin and streptomycin sulfate in a concentration-dependent manner. The average level of RNase H in serums of HBV infection group was similar to that of control group. In summary, the assay provides an alternative tool for biochemical analysis for this enzyme and indicates the feasibility of high throughput screening inhibitors of RNase H in vitro and in vivo.

Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains.

Mycobacterium smegmatis encodes several DNA repair polymerases that are adept at incorporating ribonucleotides, which raises questions about how ribonucleotides in DNA are sensed and removed. RNase H enzymes, of which M. smegmatis encodes four, are strong candidates for a surveillance role. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of M. smegmatis RnhC, a bifunctional RNase H and acid phosphatase. We report that (i) the RnhC nuclease is stringently specific for RNA:DNA hybrid duplexes; (ii) RnhC does not selectively recognize and cleave DNA-RNA or RNA-DNA junctions in duplex nucleic acid; (iii) RnhC cannot incise an embedded monoribonucleotide or diribonucleotide in duplex DNA; (iv) RnhC can incise tracts of 4 or more ribonucleotides embedded in duplex DNA, leaving two or more residual ribonucleotides at the cleaved 3'-OH end and at least one or two ribonucleotides on the 5'-PO4 end; (v) the RNase H activity is inherent in an autonomous 140-amino-acid (aa) N-terminal domain of RnhC; and (vi) the C-terminal 211-aa domain of RnhC is an autonomous acid phosphatase. The cleavage specificity of RnhC is clearly distinct from that of Escherichia coli RNase H2, which selectively incises at an RNA-DNA junction. Thus, we classify RnhC as a type I RNase H. The properties of RnhC are consistent with a role in Okazaki fragment RNA primer removal or in surveillance of oligoribonucleotide tracts embedded in DNA but not in excision repair of single misincorporated ribonucleotides. RNase H enzymes help cleanse the genome of ribonucleotides that are present either as ribotracts (e.g., RNA primers) or as single ribonucleotides embedded in duplex DNA. Mycobacterium smegmatis encodes four RNase H proteins, including RnhC, which is characterized in this study. The nucleic acid substrate and cleavage site specificities of RnhC are consistent with a role in initiating the removal of ribotracts but not in single-ribonucleotide surveillance. RnhC has a C-terminal acid phosphatase domain that is functionally autonomous of its N-terminal RNase H catalytic domain. RnhC homologs are prevalent in Actinobacteria.

Role of RNase H1 in DNA repair: removal of single ribonucleotide misincorporated into DNA in collaboration with RNase H2

Several RNases H1 cleave the RNA-DNA junction of Okazaki fragment-like RNA-DNA/DNA substrate. This activity, termed 3’-junction ribonuclease (3’-JRNase) activity, is different from the 5’-JRNase activity of RNase H2 that cleaves the 5’-side of the ribonucleotide of the RNA-DNA junction and is required to initiate the ribonucleotide excision repair pathway. To examine whether RNase H1 exhibits 3’-JRNase activity for dsDNA containing a single ribonucleotide and can remove this ribonucleotide in collaboration with RNase H2, cleavage of a DNA8-RNA1-DNA9/DNA18 substrate with E. coli RNase H1 and H2 was analyzed. This substrate was cleaved by E. coli RNase H1 at the (5’)RNA-DNA(3’) junction, regardless of whether it was cleaved by E. coli RNase H2 at the (5’)DNA-RNA(3’) junction in advance or not. Likewise, this substrate was cleaved by E. coli RNase H2 at the (5’)DNA-RNA(3’) junction, regardless of whether it was cleaved by E. coli RNase H1 at the (5’)RNA-DNA(3’) junction in advance or not. When this substrate was cleaved by a mixture of E. coli RNases H1 and H2, the ribonucleotide was removed from the substrate. We propose that RNase H1 is involved in the excision of single ribonucleotides misincorporated into DNA in collaboration with RNase H2.

Crystal structure of RNase H3-substrate complex reveals parallel evolution of RNA/DNA hybrid recognition.

RNases H participate in the replication and maintenance of genomic DNA. RNase H1 cleaves the RNA strand of RNA/DNA hybrids, and RNase H2 in addition hydrolyzes the RNA residue of RNA–DNA junctions. RNase H3 is structurally closely related to RNases H2, but its biochemical properties are similar to type 1 enzymes. Its unique N-terminal substrate-binding domain (N-domain) is related to TATA-binding protein. Here, we report the first crystal structure of RNase H3 in complex with its RNA/DNA substrate. Just like RNases H1, type 3 enzyme recognizes the 2′-OH groups of the RNA strand and detects the DNA strand by binding a phosphate group and inducing B-form conformation. Moreover, the N-domain recognizes RNA and DNA in a manner that is highly similar to the hybrid-binding domain of RNases H1. Our structure demonstrates a remarkable example of parallel evolution of the elements used in the specific recognition of RNA and DNA.

Aprataxin resolves adenylated RNA–DNA junctions to maintain genome integrity

Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions employed by ATP-dependent DNA ligases1,2. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5′-adenylated (5′-AMP) DNA lesions3–6 (Fig. 1a). Aprataxin (Aptx) reverses DNA-adenylation but the context for deadenylation repair is unclear. Here we examine the importance of Aptx to RNaseH2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5′-ends containing a ribose characteristic of RNaseH2 incision. Aptx efficiently repairs adenylated RNA-DNA, and acting in an RNA-DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure-function studies of human Aptx/RNA-DNA/AMP/Zn complexes define a mechanism for detecting and reversing adenylation at RNA-DNA junctions. This involves A-form RNA-binding, proper protein folding and conformational changes, all of which are impacted by heritable APTX mutations in Ataxia with Oculomotor Apraxia 1 (AOA1). Together, these results suggest that accumulation of adenylated RNA-DNA may contribute to neurological disease.

Real time monitoring of junction ribonuclease activity of RNase H using chimeric molecular beacons

As a highly conserved damage repair protein, except for hydrolysis of DNA-RNA heteroduplex endonucleolytically, RNase H can cleave RNA-DNA junctions in Okazaki fragment processing through its junction ribonuclease (JRNase) activity. We report here a real time fluorescence method for detecting JRNase activity of RNase H with high accuracy by applying chimeric molecular beacons as substrates. The detection limit of E. coli RNase H is 0.5 U ml(-1). The Km and kcat are 20 nM and 0.6 s(-1), respectively. In addition, we used the method to investigate the effect of chemical drugs on the enzyme and found that both penicillin and streptomycin sulfate inhibit its activity with the IC50 values of 0.2 and 0.07 mM, respectively. Finally, we applied the method to reliably detect the JRNase level in tumor cells. In summary, these data indicate that the simple, rapid and sensitive method can be hopefully applied for high-throughput detection of samples and drug screening in vitro.

RecFOR Proteins Target RecA Protein to a DNA Gap with Either DNA or RNA at the 5′ Terminus: IMPLICATION FOR REPAIR OF STALLED REPLICATION FORKS

The repair of single-stranded gaps in duplex DNA by homologous recombination requires the proteins of the RecF pathway. The assembly of RecA protein onto gapped DNA (gDNA) that is complexed with the single-stranded DNA-binding protein is accelerated by the RecF, RecO, and RecR (RecFOR) proteins. Here, we show the RecFOR proteins specifically target RecA protein to gDNA even in the presence of a thousand-fold excess of single-stranded DNA (ssDNA). The binding constant of RecF protein, in the presence of the RecOR proteins, to the junction of ssDNA and dsDNA within a gap is 1-2 nm, suggesting that a few RecF molecules in the cell are sufficient to recognize gDNA. We also found that the nucleation of a RecA filament on gDNA in the presence of the RecFOR proteins occurs at a faster rate than filament elongation, resulting in a RecA nucleoprotein filament on ssDNA for 1000-2000 nucleotides downstream (5' → 3') of the junction with duplex DNA. Thus, RecA loading by RecFOR is localized to a region close to a junction. RecFOR proteins also recognize RNA at the 5'-end of an RNA-DNA junction within an ssDNA gap, which is compatible with their role in the repair of lagging strand gaps at stalled replication forks.

Involvement of Ser94 in RNase HIII from Chlamydophila pneumoniae in the recognition of a single ribonucleotide misincorporated into double-stranded DNA

We recently provided the first report that RNase HIII can cleave a DNA-rN1-DNA/DNA substrate (rN1, one ribonucleotide) in vitro. In the present study, mutagenesis analyses and molecular dynamics (MD) simulations were performed on RNase HIII from Chlamydophila pneumoniae AR39 (CpRNase HIII). Our results elucidate the mechanism of ribonucleotide recognition employed by CpRNase HIII, indicating that the G95/K96/G97 motif of CpRNase HIII represents the main surface interacting with single ribonucleotides, in a manner similar to that of the GR(K)G motif of RNase HIIs. However, CpRNase HIII lacks the specific tyrosine required for RNase HII to recognize single ribonucleotides in double-stranded DNA (dsDNA). Interestingly, MD shows that Ser94 of CpRNase HIII forms a stable hydrogen bond with the deoxyribonucleotide at the (5')RNA–DNA(3') junction, moving this nucleotide away from the chimeric ribonucleotide. This movement appears to deform the nucleic acid backbone at the RNA–DNA junction and allows the ribonucleotide to interact with the GKG motif. Based on the inferences drawn from MD simulations, biochemical results indicated that Ser94 was necessary for catalytic activity on the DNA-rN1-DNA/DNA substrate; mutant S94V could bind this substrate but exhibited no cleavage. Mismatches opposite the single ribonucleotide misincorporated in dsDNA inhibited cleavage by CpRNase HIII to varying degrees but did not interfere with CpRNase/substrate binding. Further MD results implied that mismatches impair the interaction between Ser94 and the deoxyribonucleotide at the RNA–DNA junction. Consequently, recognition of the misincorporated ribonucleotide was disturbed. Our results may help elucidate the distinct substrate-recognition properties of different RNase Hs.

Study of Helicase Activity of the Ribosome using Single-Molecule FRET

Secondary and tertiary structures of mRNA may become barriers to the ribosome during translation. Previous studies have shown that the ribosome itself is capable of opening the base-paring of mRNA. More recently, by using optical tweezers, Qu et al. have shown that this unwinding process involves two kinds of active mechanisms, in which the ribosome destabilizes and mechanically unwinds the encountered junctions on mRNA. However, detailed interaction between the ribosome and mRNA at the junction remains to be elucidated. In this study, we aim to utilize TIRF (total internal reflection fluorescence) microscopy to detect FRET signals at single-molecule level, so that we can observe the stepwise unwinding process during translocation in real-time. We have purified all the translation factors as well as the ribosome required for this system from Escherichia coli. We have also constructed a template to mimic RNA duplex structures by pairing a Cy5-labeled DNA oligonucleotide to a Cy3-labeled mRNA. Then, the duplex will be mixed with the defined in vitro translation system that allows us to control each translocation step of the ribosome by adding one unique aminoacyl-tRNA at a time. We expect the FRET efficiency of the dye pair will fluctuate as the ribosome translocates through the RNA-DNA junctions.

The Structural and Biochemical Characterization of Human RNase H2 Complex Reveals the Molecular Basis for Substrate Recognition and Aicardi-Goutières Syndrome Defects

RNase H2 cleaves RNA sequences that are part of RNA/DNA hybrids or that are incorporated into DNA, thus, preventing genomic instability and the accumulation of aberrant nucleic acid, which in humans induces Aicardi-Goutières syndrome, a severe autoimmune disorder. The 3.1 Å crystal structure of human RNase H2 presented here allowed us to map the positions of all 29 mutations found in Aicardi-Goutières syndrome patients, several of which were not visible in the previously reported mouse RNase H2. We propose the possible effects of these mutations on the protein stability and function. Bacterial and eukaryotic RNases H2 differ in composition and substrate specificity. Bacterial RNases H2 are monomeric proteins and homologs of the eukaryotic RNases H2 catalytic subunit, which in addition possesses two accessory proteins. The eukaryotic RNase H2 heterotrimeric complex recognizes RNA/DNA hybrids and (5′)RNA-DNA(3′)/DNA junction hybrids as substrates with similar efficiency, whereas bacterial RNases H2 are highly specialized in the recognition of the (5′)RNA-DNA(3′) junction and very poorly cleave RNA/DNA hybrids in the presence of Mg2+ ions. Using the crystal structure of the Thermotoga maritima RNase H2-substrate complex, we modeled the human RNase H2-substrate complex and verified the model by mutational analysis. Our model indicates that the difference in substrate preference stems from the different position of the crucial tyrosine residue involved in substrate binding and recognition.

Crystal Structures of RNase H2 in Complex with Nucleic Acid Reveal the Mechanism of RNA-DNA Junction Recognition and Cleavage

SummaryTwo classes of RNase H hydrolyze RNA of RNA/DNA hybrids. In contrast to RNase H1 that requires four ribonucleotides for cleavage, RNase H2 can nick duplex DNAs containing a single ribonucleotide, suggesting different in vivo substrates. We report here the crystal structures of a type 2 RNase H in complex with substrates containing a (5′)RNA-DNA(3′) junction. They revealed a unique mechanism of recognition and substrate-assisted cleavage. A conserved tyrosine residue distorts the nucleic acid at the junction, allowing the substrate to function in catalysis by participating in coordination of the active site metal ion. The biochemical and structural properties of RNase H2 explain the preference of the enzyme for junction substrates and establish the structural and mechanistic differences with RNase H1. Junction recognition is important for the removal of RNA embedded in DNA and may play an important role in DNA replication and repair.

Structure-Specific Nuclease Activities of Pyrococcus abyssi RNase HII

Faithful DNA replication involves the removal of RNA residues from genomic DNA prior to the ligation of nascent DNA fragments in all living organisms. Because the physiological roles of archaeal type 2 RNase H are not fully understood, the substrate structure requirements for the detection of RNase H activity need further clarification. Biochemical characterization of a single RNase H detected within the genome of Pyrococcus abyssi showed that this type 2 RNase H is an Mg- and alkaline pH-dependent enzyme. PabRNase HII showed RNase activity and acted as a specific endonuclease on RNA-DNA/DNA duplexes. This specific cleavage, 1 nucleotide upstream of the RNA-DNA junction, occurred on a substrate in which RNA initiators had to be fully annealed to the cDNA template. On the other hand, a 5' RNA flap Okazaki fragment intermediate impaired PabRNase HII endonuclease activity. Furthermore, introduction of mismatches into the RNA portion near the RNA-DNA junction decreased both the specificity and the efficiency of cleavage by PabRNase HII. Additionally, PabRNase HII could cleave a single ribonucleotide embedded in a double-stranded DNA. Our data revealed PabRNase HII as a dual-function enzyme likely required for the completion of DNA replication and DNA repair.

Junction ribonuclease activity specified in RNases HII/2

Junction ribonuclease (JRNase) recognizes the transition from RNA to DNA of an RNA-DNA/DNA hybrid, such as an Okazaki fragment, and cleaves it, leaving a mono-ribonucleotide at the 5' terminus of the RNA-DNA junction. Although this JRNase activity was originally reported in calf RNase H2, some other RNases H have recently been suggested to possess it. This paper shows that these enzymes can also cleave an RNA-DNA/RNA heteroduplex in a manner similar to the RNA-DNA/DNA substrate. The cleavage site of the RNA-DNA/RNA substrate corresponds to the RNA/RNA duplex region, indicating that the cleavage activity cannot be categorized as RNase H activity, which specifically cleaves an RNA strand of an RNA/DNA hybrid. Examination of several RNases H with respect to JRNase activity suggested that the activity is only found in RNase HII orthologs. Therefore, RNases HIII, which are RNase HII paralogs, are distinguished from RNases HII by the absence of JRNase activity. Whether a substrate can be targeted by JRNase activity would depend only on whether or not an RNA-DNA junction consisting of one ribonucleotide and one deoxyribonucleotide is included in the duplex. In addition, although the activity has been reported not to occur on completely single-stranded RNA-DNA, it can recognize a single-stranded RNA-DNA junction if a double-stranded region is located adjacent to the junction.

Junction ribonuclease: a ribonuclease HII orthologue from Thermus thermophilus HB8 prefers the RNA–DNA junction to the RNA/DNA heteroduplex

The genome of an extremely thermophilic bacterium, Thermus thermophilus HB8, contains a single ORF (open reading frame) encoding an RNase-HII-like sequence. Despite the presence of significant amino acid sequence identities with RNase (ribonuclease) HII enzymes, the ORF TTHA0198 could not suppress the temperature-sensitive growth defect of an RNase-H-deficient Escherichia coli mutant and the purified recombinant protein could not cleave an RNA strand of an RNA/DNA heteroduplex, suggesting that the TTHA0198 exhibited no RNase H activity both in vivo and in vitro. When oligomeric RNA-DNA/DNAs were used as a mimic substrate for Okazaki fragments, however, the protein cleaved them only at the 5' side of the last ribonucleotide at the RNA-DNA junction. In fact, the TTHA0198 protein prefers the RNA-DNA junction to the RNA/DNA hybrid. We have referred to this activity as JRNase (junction RNase) activity, which recognizes an RNA-DNA junction of the RNA-DNA/DNA heteroduplex and cleaves it leaving a mono-ribonucleotide at the 5' terminus of the RNA-DNA junction. E. coli and Deinococcus radiodurans RNases HII also cleaved the RNA-DNA/DNA substrates at the same site with a different metal-ion preference from that for RNase H activity, implying that the enzymes have JRNase activity as well as RNase H activity. The specialization in the JRNase activity of the RNase HII orthologue from T. thermophilus HB8 (Tth-JRNase) suggests that the JRNase activity of RNase HII enzymes might be independent of the RNase H activity.