The RNase H-like superfamily: new members, comparative structural analysis and evolutionary classification.

Ribonuclease H-like (RNHL) superfamily, also called the retroviral integrase superfamily, groups together numerous enzymes involved in nucleic acid metabolism and implicated in many biological processes, including replication, homologous recombination, DNA repair, transposition and RNA interference. The RNHL superfamily proteins show extensive divergence of sequences and structures. We conducted database searches to identify members of the RNHL superfamily (including those previously unknown), yielding >60 000 unique domain sequences. Our analysis led to the identification of new RNHL superfamily members, such as RRXRR (PF14239), DUF460 (PF04312, COG2433), DUF3010 (PF11215), DUF429 (PF04250 and COG2410, COG4328, COG4923), DUF1092 (PF06485), COG5558, OrfB_IS605 (PF01385, COG0675) and Peptidase_A17 (PF05380). Based on the clustering analysis we grouped all identified RNHL domain sequences into 152 families. Phylogenetic studies revealed relationships between these families, and suggested a possible history of the evolution of RNHL fold and its active site. Our results revealed clear division of the RNHL superfamily into exonucleases and endonucleases. Structural analyses of features characteristic for particular groups revealed a correlation between the orientation of the C-terminal helix with the exonuclease/endonuclease function and the architecture of the active site. Our analysis provides a comprehensive picture of sequence-structure-function relationships in the RNHL superfamily that may guide functional studies of the previously uncharacterized protein families.

Cloning of the RNase H genes from a metagenomic DNA library: identification of a new type 1 RNase H without a typical active-site motif.

AIMS: The study aimed to combine a metagenomics approach with complementary genetics to identify novel bacterial genes with orthologous functions, with the identification of novel RNase H genes as a test case. METHODS AND RESULTS: A metagenomic DNA library was prepared from leaf-and-branch compost and used to screen for the RNase H genes by their abilities to complement the temperature-sensitive growth phenotype of the rnhA mutant Escherichia coli strain MIC3001. Determination of the nucleotide sequences of the cloned DNA fragments allowed us to identify 12 different genes encoding type 1 RNases H. Eleven of them encode novel RNases H, which show 40-72% amino acid sequence identities to those available from database. One of them lacks a typical DEDD/E active-site motif, which is almost fully conserved in various RNases H. CONCLUSIONS: Functional screening of environmental DNA without cultivation of microbes is a useful procedure to isolate novel RNase H genes. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the identified RNase H genes had no sequence similarity to a previously assumed conserved motif, suggesting multiple catalytic mechanisms exist. This test case illustrates that metagenomics combined with complementary genetics can identify novel genes that are orthologous without sequence similarity to those from cultivated bacteria.

Identification of the gene encoding a type 1 RNase H with an N-terminal double-stranded RNA binding domain from a psychrotrophic bacterium.

The gene encoding a bacterial type 1 RNase H, termed RBD-RNase HI, was cloned from the psychrotrophic bacterium Shewanella sp. SIB1, overproduced in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RBD-RNase HI consists of 262 amino acid residues and shows amino acid sequence identities of 26% to SIB1 RNase HI, 17% to E. coli RNase HI, and 32% to human RNase H1. SIB1 RBD-RNase HI has a double-stranded RNA binding domain (RBD) at the N-terminus, which is commonly present at the N-termini of eukaryotic type 1 RNases H. Gel mobility shift assay indicated that this domain binds to an RNA/DNA hybrid in an isolated form, suggesting that this domain is involved in substrate binding. SIB1 RBD-RNase HI exhibited the enzymatic activity both in vitro and in vivo. Its optimum pH and metal ion requirement were similar to those of SIB1 RNase HI, E. coli RNase HI, and human RNase H1. The specific activity of SIB1 RBD-RNase HI was comparable to that of E. coli RNase HI and was much higher than those of SIB1 RNase HI and human RNase H1. SIB1 RBD-RNase HI showed poor cleavage-site specificity for oligomeric substrates. SIB1 RBD-RNase HI was less stable than E. coli RNase HI but was as stable as human RNase H1. Database searches indicate that several bacteria and archaea contain an RBD-RNase HI. This is the first report on the biochemical characterization of RBD-RNase HI.

Redesignation of the RNase D activity associated with retroviral reverse transcriptase as RNase H.

In the presence of Mn2+, reverse transcriptase of both human immunodeficiency virus and murine leukemia virus hydrolyzes duplex RNA. However, designating this novel activity RNase D conflicts with Escherichia coli RNase D, which participates in tRNA processing. On the basis of its location in the RNase H domain, we propose that this novel retroviral activity be redesignated RNase H*.

Molecular cloning and expression of cDNA for human RNase H.

We have cloned, expressed, and purified to electrophoretic homogeneity a human RNase H. The enzyme has a molecular weight of 32 kDa, is Mg2+ dependent, and is inhibited by Mn2+ and N-ethylmaleimide. Its molecular weight and cleavage characteristics are consistent with type 2 human RNase H. The human RNase H we have cloned is highly homologous to Escherichia coli RNase HI (33.6% amino acid identity) and to other RNase H enzymes homologous to E. coli RNase HI. The enzyme is encoded by a single gene that is at least 10 kb in length and is expressed ubiquitously in human cells and tissues.

Sequence and comparative structural analysis of the murine leukaemia virus amphotropic strain 4070A RNase H domain.

The sequence of a 900-nucleotide segment (encoding part of the reverse transcriptase, including the entire RNase H domain) of the pol gene of the murine leukaemia virus (MLV) amphotropic strain 4070A is presented. Alignment of the inferred 4070A RNase H amino acid sequence (157 residues) with other MLV RNase H sequences revealed only minor differences compared with the divergence between other retroviral and prokaryotic or eukaryotic RNase H sequences. Only 10 residues were invariant across the entire sample set, but secondary structure predictions for the enzymes from E. coli, yeast, human liver and diverse retroviruses (HIV, Rous sarcoma virus, foamy viruses) supported, in every case, the five beta-strands (1 to 5) and four or five alpha-helices (A, B/C, D, E) that have been identified by crystallography in the RNase H domain of HIV-1 reverse transcriptase and in E. coli RNase H. In the case of MLV, analysis of the RNase H domain sequences inferred from 10 different strains (including the amphotropic 4070A) predicted all five alpha-helices (A-E), as well as beta-strands 4 and 5. However, the N-terminal segment (residues 1-40) was predicted, without exception and with high probability, to fold uniquely into one (or two adjacent) alpha-helix(es) encompassing residues 13-37, instead of the three beta-strands known to exist in the HIV-1 and E. coli enzymes. The unerring consistency between the known and predicted structures of the HIV-1 and E. coli enzymes, and the prediction of the same structural elements (including beta-strands 1-3 within the N-terminal segment) for all other (non-MLV) RNase H proteins examined in this study, suggests that the N-terminal segment of the MLV RNase H domain assumes a conformation distinct from that of other retroviral and cellular RNase H molecules. An additional (sixth) beta-strand was also predicted uniquely within the C-terminal region of foamy virus RNase H domains.