PCNA directs type 2 RNase H activity on DNA replication and repair substrates

Ribonuclease H2 is the major nuclear enzyme degrading cellular RNA/DNA hybrids in eukaryotes and the sole nuclease known to be able to hydrolyze ribonucleotides misincorporated during genomic replication. Mutation in RNASEH2 causes Aicardi-Goutières syndrome, an auto-inflammatory disorder that may arise from nucleic acid byproducts generated during DNA replication. Here, we report the crystal structures of Archaeoglobus fulgidus RNase HII in complex with PCNA, and human PCNA bound to a C-terminal peptide of RNASEH2B. In the archaeal structure, three binding modes are observed as the enzyme rotates about a flexible hinge while anchored to PCNA by its PIP-box motif. PCNA binding promotes RNase HII activity in a hinge-dependent manner. It enhances both cleavage of ribonucleotides misincorporated in DNA duplexes, and the comprehensive hydrolysis of RNA primers formed during Okazaki fragment maturation. In addition, PCNA imposes strand specificity on enzyme function, and by localizing RNase H2 and not RNase H1 to nuclear replication foci in vivo it ensures that RNase H2 is the dominant RNase H activity during nuclear replication. Our findings provide insights into how type 2 RNase H activity is directed during genome replication and repair, and suggest a mechanism by which RNase H2 may suppress generation of immunostimulatory nucleic acids.     

Transcriptional role in DNA replication: degradation of RNA primer during DNA synthesis

DNA synthesis catalyzed in vitro by E. coli DNA polymeraseI in the presence of single stranded fd DNA or poly (dT) as template is stimulated by RNA primers. When poly(dT) fully or partially saturated with polyriboadenylic acid strands is used as template - primer, DNA synthesis proceeds with concomitant degradation of the ribostrands to 5′-adenosine monophosphate. The fragment of DNA polymerase lacking the 5′→3′ exonuclease shows comparable RNA primer dependency but reduced efficiency for the degradation of the RNA primer from the 5′-end.     

Saccharomyces cerevisiae RNase H(35) functions in RNA primer removal during lagging-strand DNA synthesis, most efficiently in cooperation with Rad27 nuclease

Correct removal of RNA primers of Okazaki fragments during lagging-strand DNA synthesis is a critical process for the maintenance of genome integrity. Disturbance of this process has severe mutagenic consequences and could contribute to the development of cancer. The role of the mammalian nucleases RNase HI and FEN-1 in RNA primer removal has been substantiated by several studies. Recently, RNase H(35), the Saccharomyces cerevisiae homologue of mammalian RNase HI, was identified and its possible role in DNA replication was proposed (P. Frank, C. Braunshofer-Reiter, and U. Wintersberger, FEBS Lett. 421:23-26, 1998). This led to the possibility of moving to the genetically powerful yeast system for studying the homologues of RNase HI and FEN-1, i.e., RNase H(35) and Rad27p, respectively. In this study, we have biochemically defined the substrate specificities and the cooperative as well as independent cleavage mechanisms of S. cerevisiae RNase H(35) and Rad27 nuclease by using Okazaki fragment model substrates. We have also determined the additive and compensatory pathological effects of gene deletion and overexpression of these two enzymes. Furthermore, the mutagenic consequences of the nuclease deficiencies have been analyzed. Based on our findings, we suggest that three alternative RNA primer removal pathways of different efficiencies involve RNase H(35) and Rad27 nucleases in yeast.     

Use of RNase H and primer extension to analyze RNA splicing.

A new method for the characterization of pre-mRNA splicing products is presented. In this method RNA molecules are hybridized to an oligodeoxynucleotide complementary to exon sequences upstream of a given 5' splice site, and the RNA strands of the resulting RNA:DNA hybrids are cleaved by RNase H. The cleaved RNAs are then subjected to primer extension using a 32P-labelled primer complementary to exon sequences downstream of an appropriate 3' splice site. Since the primer extension products all terminate at the site of RNase H cleavage, their lengths are indicative of the splice sites utilized. The method simplifies the study of the processing of complex pre-mRNAs by allowing the splicing events between any two exons to be analyzed. We have used this approach to characterize the RNAs generated by expression of the rat tropomyosin 1 (Tm 1) gene in various rat tissues and in cultured cells after transient transfection. The results demonstrate that this method is suitable for the analysis of alternative RNA processing in vivo.     

Function of RNase H in DNA replication revealed by RNase H defective mutants of Escherichia coli

Summary Escherichia coli rnh mutants were isolated using localized mutagenesis and selective measurements of RNase H activity in mutagenized cell extracts with [³H]poly(rC)·poly(dG) as substrate. RNase H activity in extracts of one mutant, ON152 (rnh-91), was undetectable (less than 0.05% of that of wild-type cells). This mutant formed small colonies at 43 °C. At this temperature, accumulation of nascent fragments was more prominent in the rnh-91·polA4113 double mutant than in the polA4113 mutant; however, no accumulation was found in the rnh single mutant at 43° C. Unlike the 1–3 nucleotide primer RNA found on nascent fragments of polA4113 cells, primers from the rnh-91·polA4113 cells ranged from one to about ten bases. These results suggest that the 53 exonuclease activity of DNA polymerase I plays a major role in removal of primer RNA and that RNase H functions in an auxiliary role, excising the 5-portion of longer primers.The rnh mutant supports replication of ColE1-type plasmids. A possible mechanism of replication of such plasmids in rnh mutants and a role of RNase H in the initiation of chromosomal replication are discussed.     

RNase H-defective mutants of Escherichia coli: a possible discriminatory role of RNase H in initiation of DNA replication

Summary Mutants of Escherichia coli completely deficient in RNase H activity were isolated by inserting transposon Tn3 into the structural gene for RNase H, rnh, and its promoter. These rnh ^- mutants exhibited the following phenotypes; (1) the mutants grew fairly normally, (2) rnh ^- cells could be transformed with ColE1 derivative plasmids, pBR322 and pML21, though the plasmids were relatively unstable, under non selective conditions, (3) rnh ^- mutations partially suppressed the temperature-sensitive phenotype of plasmid pSC301, a DNA replication initiation mutant derived from pSC101, (4) rnh ^- mutations suppressed the temperature-sensitive growth character of dnaA ^ts mutant, (5) rnh ^- cells showed continued DNA synthesis in the presence of chloramphenicol (stable DNA replication). Based on these findings we propose a model for a role of RNase H in the initiation of chromosomal DNA replication. We suggest that two types of RNA primers for initiation of DNA replication are synthesized in a dnaA/oriC-dependent and-independent manner and that only the dnaA/oriC-dependent primer is involved in the normal DNA replication since the dnaA/oriC independent primer is selectively degraded by RNase H.Abbreviations AP^r ampicillin-resistant - kb kilobase pair(s) - NEM N-ethyl maleimide - Ts temperature-sensitive     

RNase H and replication of ColE1 DNA in Escherichia coli

Amber mutations within the rnh (RNase H) gene of Escherichia coli K-12 were isolated by selecting for bacteria capable of replicating in a sup+ background replication-defective cer-6 mutant of the ColE1 replicon. The cer-6 mutation is an alteration of one base pair located 160 nucleotides upstream of the unique replication origin of this plasmid. Subsequently, we determined the DNA alterations present within these mutants. ColE1 DNA replicated in rnh(Am) recA cells, indicating that (i) RNase H, which has been shown to be absolutely required for in vitro initiation of ColE1 DNA replication, is dispensable in vivo, and (ii) ColE1 replication in the absence of RNase H is not dependent on "stable DNA replication," which has been reported to be an alternative mode of chromosomal DNA replication. Another class of bacterial mutations was also isolated. These mutations, named herB, suppressed cer-6 replication in rnh+ bacteria. herB mutations mapped close to the polA gene on the E. coli chromosome and increased the activity of DNA polymerase I. These findings suggest that when the DNA polymerase I has an opportunity to initiate DNA synthesis before RNase H acts, the replication-defective cer-6 mutant or the wild-type ColE1 replicates in E. coli.     

Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD

Human RNase H1 contains an N-terminal domain known as dsRHbd for binding both dsRNA and RNA/DNA hybrid. We find that dsRHbd binds preferentially to RNA/DNA hybrids by over 25-fold and rename it as hybrid binding domain (HBD). The crystal structure of HBD complexed with a 12 bp RNA/DNA hybrid reveals that the RNA strand is recognized by a protein loop, which forms hydrogen bonds with the 2'-OH groups. The DNA interface is highly specific and contains polar residues that interact with the phosphate groups and an aromatic patch that appears selective for binding deoxyriboses. HBD is unique relative to non-sequence-specific dsDNA- and dsRNA-binding domains because it does not use positive dipoles of alpha-helices for nucleic acid binding. Characterization of full-length enzymes with defective HBDs indicates that this domain dramatically enhances both the specific activity and processivity of RNase H1. Similar activity enhancement by small substrate-binding domains linked to the catalytic domain likely occurs in other nucleic acid enzymes.     

Sequence-specific cleavage of RNA using chimeric DNA splints and RNase H

To cleave RNA molecules using E. coli RNase H in a site-specific manner, a short oligodeoxyribonucleotide (3-5 mer) linked with oligo(2'-O-methyl)ribonucleotide(s) was designed to be used as a DNA splint. Our model experiments with ribooligomer the splint duplexes (9 mers) and RNase H demonstrated that a tetradeoxynucleotide cluster seems to be sufficient for the enzyme recognition and the short DNA-containing splint directs a unique cleavage of RNA by RNase H. The method could be applied to longer ribooligonucleotide substrates. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.     

Targeting degradation of RNA by RNase H using DNA hairpins

RNase H degradation of two 15 nt RNA target sites was examined in the presence of hairpin DNAs with a 5 nt loop and a 10 bp stem or single-stranded 15 nt DNAs. One target site was a segment of a 79 nt RNA, and the other was part of a 53 nt RNA. Secondary structure predictions indicate that the 53 nt RNA target site is entirely single stranded, while a portion of the 79 nt RNA target site forms an intramolecular duplex. Less RNase H and DNA were needed to cleave the 53 nt RNA target site than the less accessible 79 nt RNA site. The hairpin DNAs had their 5 nt loop and 3' side of the stem fully complementary to the target sites or had sequence changes that produced one to nine mismatched pairs. T(m) values ranged from 57 to 80 degrees C. The stability of the hairpin DNAs relative to the stability of their corresponding RNA-DNA hybrids influenced the extent of RNase H degradation at 37 degrees C. Under the assay conditions employed, the amount of degradation directed by the hairpin DNAs was correlated with their predicted DeltaG(o) (37) of binding to the RNA targets. A DNA hairpin with one mismatch to the target site of the 79 nt RNA did not induce degradation under conditions where fully complementary DNA hairpins produced 50-80% degradation. The in vitro results indicate that DNA hairpins can enhance the stringency of RNase H targeted degradation of the RNA sites.     

NMR structure of an alpha-L-LNA:RNA hybrid: structural implications for RNase H recognition

Alpha-L-LNA (alpha-L-ribo configured locked nucleic acid) is a nucleotide analogue that raises the thermostability of nucleic acid duplexes by up to approximately 4 degrees C per inclusion. We have determined the NMR structure of a nonamer alpha-L-LNA:RNA hybrid with three alpha-L-LNA modifications. The geometry of this hybrid is intermediate between A- and B-type, all nucleobases partake in Watson-Crick base pairing and base stacking, and the global structure is very similar to that of the corresponding unmodified hybrid. The sugar-phosphate backbone is rearranged in the vicinity of the modified nucleotides. As a consequence, the phosphate groups following the modified nucleotides are rotated into the minor groove. It is interesting that the alpha-L-LNA:RNA hybrid, which has an elevation in melting temperature of 17 degrees C relative to the corresponding DNA:RNA hybrid, retains the global structure of this hybrid. To our knowledge, this is the first example of such a substantial increase in melting temperature of a nucleic acid analogue that does not act as an N-type (RNA) mimic. alpha-L-LNA:RNA hybrids are recognised by RNase H with subsequent cleavage of the RNA strand, albeit with slow rates. We attempt to rationalise this impaired enzyme activity from the rearrangement of the sugar-phosphate backbone of the alpha-L-LNA:RNA hybrid.     

Insights into RNA/DNA hybrid recognition and processing by RNase H from the crystal structure of a non-specific enzyme-dsDNA complex

Ribonuclease HI (RNase H) is a member of the nucleotidyl-transferase superfamily and endo-nucleolytically cleaves the RNA portion in RNA/DNA hybrids and removes RNA primers from Okazaki fragments. The enzyme also binds RNA and DNA duplexes but is unable to cleave either. Three-dimensional structures of bacterial and human RNase H catalytic domains bound to RNA/DNA hybrids have revealed the basis for substrate recognition and the mechanism of cleavage. In order to visualize the enzyme’s interactions with duplex DNA and to establish the structural differences that afford tighter binding to RNA/DNA hybrids relative to dsDNA, we have determined the crystal structure of Bacillus halodurans RNase H in complex with the B-form DNA duplex [d(CGCGAATTCGCG)]2. The structure demonstrates that the inability of the enzyme to cleave DNA is due to the deviating curvature of the DNA strand relative to the substrate RNA strand and the absence of Mg2+ at the active site. A subset of amino acids engaged in contacts to RNA 2′-hydroxyl groups in the substrate complex instead bind to bridging or non-bridging phosphodiester oxygens in the complex with dsDNA. Qualitative comparison of the enzyme’s interactions with the substrate and inhibitor duplexes is consistent with the reduced binding affinity for the latter and sheds light on determinants of RNase H binding and cleavage specificity.     

Structural basis of the RNase H1 activity on stereo regular borano phosphonate DNA/RNA hybrids

Numerous DNA chemistries for improving oligodeoxynucleotide (ODN)-based RNA targeting have been explored. The majority of the modifications render the ODN/RNA target insensitive to RNase H1. Borano phosphonate ODN's are among the few modifications that are tolerated by RNase H1. To understand the effect of the stereochemistry of the BH(3) modification on the nucleic acid structure and RNase H1 enzyme activity, we have investigated two DNA/RNA hybrids containing either a R(P) or S(P) BH(3) modification by nuclear magnetic resonance (NMR) spectroscopy. T(M) studies show that the stabilities of R(P) and S(P) modified DNA/RNA hybrids are essentially identical (313.8 K) and similar to that of an unmodified control (312.9 K). The similarity is also reflected in the imino proton spectra. To characterize such similar structures, we used a large number of NMR restraints (including dipolar couplings and backbone torsion angles) to determine structural features that were important for RNase H1 activity. The final NMR structures exhibit excellent agreement with the data (total R(x) values of <6%) with helical properties between those of an A and B helix. Subtle backbone variations are observed in the DNA near the modification, while the RNA strands are relatively unperturbed. In the case of the S(P) modification, for which more perturbations are recorded, a slightly narrower minor groove is also obtained. Unique NOE base contacts localize the S(P) BH(3) group in the major groove while the R(P) BH(3) group points away from the DNA. However, this creates a potential clash of the R(P) BH(3) groups with important RNase H1 residues in a complex, while the S(P) BH(3) groups could be tolerated. We therefore predict that on the basis of our NMR structures a fully R(P) BH(3) DNA/RNA hybrid would not be a substrate for RNase H1.     

Site-specific cleavage of MS2 RNA by a thermostable DNA-linked RNase H

A series of DNA-linked RNases H, in which the 15-mer DNA is cross-linked to the Thermus thermophilus RNase HI (TRNH) variants at positions 135, 136, 137 and 138, were constructed and analyzed for their abilities to cleave the complementary 15-mer RNA. Of these, that with the DNA adduct at position 135 most efficiently cleaved the RNA substrate, indicating that position 135 is the most appropriate cross-linking site among those examined. To examine whether DNA-linked RNase H also site-specifically cleaves a highly structured natural RNA, DNA-linked TRNHs with a series of DNA adducts varying in size at position 135 were constructed and analyzed for their abilities to cleave MS2 RNA. These DNA adducts were designed such that DNA-linked enzymes cleave MS2 RNA at a loop around residue 2790. Of the four DNA-linked TRNHs with the 8-, 12-, 16- and 20-mer DNA adducts, only that with the 16-mer DNA adduct efficiently and site-specifically cleaved MS2 RNA. Primer extension revealed that this DNA-linked TRNH cleaved MS2 RNA within the target sequence.     

RNase P RNA mediated cleavage: substrate recognition and catalysis

The universally conserved endoribonuclease P consists of one RNA subunit and, depending on its origin, a variable number of protein subunits. RNase P is involved in the processing of a large variety of substrates in the cell, the preferred substrate being tRNA precursors. Cleavage activity does not require the presence of the protein subunit(s) in vitro. This is true for both prokaryotic and eukaryotic RNase P RNA suggesting that the RNA based catalytic activity has been preserved during evolution. Progress has been made in our understanding of the contribution of residues and chemical groups both in the substrate as well as in RNase P RNA to substrate binding and catalysis. Moreover, we have access to two crystal structures of bacterial RNase P RNA but we still lack the structure of RNase P RNA in complex with its substrate and/or the protein subunit. Nevertheless, these recent advancements put us in a new position to study the way and nature of interactions between in particular RNase P RNA and its substrate. In this review I will discuss various aspects of the RNA component of RNase P with an emphasis on our current understanding of the interaction between RNase P RNA and its substrate.     

Structural basis of cleavage by RNase H of hybrids of arabinonucleic acids and RNA

The origins of the substrate specificity of Escherichia coli RNase H1 (termed RNase H here), an enzyme that hydrolyzes the RNA strand of DNA-RNA hybrids, are not understood at present. Although the enzyme binds double-stranded RNA, no cleavage occurs with such duplexes [Lima, W. F., and Crooke, S. T. (1997) Biochemistry 36, 390]. Therefore, the hybrid substrates may not adopt a canonical A-form geometry. Furthermore, RNase H is exquisitely sensitive to chemical modification of the DNA strands in hybrid duplexes. This is particularly relevant to the RNase H-dependent pathway of antisense action. Thus, only very few of the modifications currently being evaluated as antisense therapeutics are tolerated by the enzyme, among them phosphorothioate DNA (PS-DNA). Recently, hybrids of RNA and arabinonucleic acid (ANA) as well as the 2'F-ANA analogue were shown to be substrates of RNase H [Damha, M. J., et al. (1998) J. Am. Chem. Soc. 120, 12976]. Using X-ray crystallography, we demonstrate here that ANA analogues, such as 2'F-ANA [Berger, I., et al. (1998) Nucleic Acids Res. 26, 2473] and [3.3.0]bicyclo-ANA (bc-ANA), may not be able to adopt sugar puckers that are compatible with pure A- or a B-form duplex geometries, but rather prefer the intermediate O4'-endo conformation. On the basis of the observed conformations of these ANA analogues in a DNA dodecamer duplex, we have modeled a duplex of an all-C3'-endo RNA strand and an all-O4'-endo 2'F-ANA strand. This duplex exhibits a minor groove width that is intermediate between that of A-form RNA and B-form DNA, a feature that may be exploited by the enzyme in differentiating between RNA duplexes and DNA-RNA hybrids. Therefore, the combination of the established structural and functional properties of ANA analogues helps settle existing controversies concerning the discrimination of substrates by RNase H. Knowlegde of the structure of an analogue that exhibits enhanced RNA affinity while not interfering with RNase H activity may prove helpful in the design of future antisense modifications.