Рибонуклеаза H ферменттерінің отбасысы

Enzyme family

Ribonuclease H (abbreviated RNase H or RNH) is a family of non sequence specific endonuclease enzymes that catalyze the cleavage of RNA in an RNA/DNA substrate via a hydrolytic mechanism. Members of the RNase H family can be found in nearly all organisms, from bacteria to archaea to eukaryotes. The family is divided into evolutionarily related groups with slightly different substrate preferences, broadly designated ribonuclease H1 and H2. The human genome encodes both H1 and H2. Human ribonuclease H2 is a heterotrimeric complex composed of three subunits, mutations in any of which are among the genetic causes of a rare disease known as Aicardi–Goutières syndrome. whereas H1 and H2 occur in all domains of life. In eukaryotes, ribonuclease H1 is involved in DNA replication of the mitochondrial genome. Both H1 and H2 are involved in genome maintenance tasks such as processing of R loop structures. RNases H have been proposed as members of an evolutionarily related superfamily encompassing other nucleases and nucleic acid processing enzymes such as retroviral integrases, DNA transposases, Holliday junction resolvases, Piwi and Argonaute proteins, various exonucleases, and the spliceosomal protein Prp8. RNases H can be broadly divided into two subtypes, H1 and H2, which for historical reasons are given Arabic numeral designations in eukaryotes and Roman numeral designations in prokaryotes. Thus the Escherichia coli RNase HI is a homolog of the Homo sapiens RNase H1. All RNases H have an active site centered on a conserved sequence motif composed of aspartate and glutamate residues, often referred to as the DEDD motif. These residues interact with catalytically required magnesium ions. While all members of the H1 group and the prokaryotic members of the H2 group function as monomers, eukaryotic H2 enzymes are obligate heterotrimers. Within the H1 group, a relationship has been identified between higher substrate binding affinity and the presence of structural elements consisting of a helix and flexible loop providing a larger and more basic substrate binding surface. The C helix has a scattered taxonomic distribution; it is present in the E. coli and human RNase H1 homologs and absent in the HIV RNase H domain, but examples of retroviral domains with C helices do exist.

Ribonuclease H enzymes cleave the phosphodiester bonds of RNA in a double stranded RNA:DNA hybrid, leaving a 3' hydroxyl and a 5' phosphate group on either end of the cut site with a two metal ion catalysis mechanism, in which two divalent cations, such as Mg2+ and Mn2+, directly participate in the catalytic function. Type 1 RNases H have prokaryotic and eukaryotic RNases H1 and retroviral RNase H. Type 2 RNases H have prokaryotic and eukaryotic RNases H2 and bacterial RNase H3. These RNases H exist in a monomeric form, except for eukaryotic RNases H2, which exist in a heterotrimeric form. RNase H1 and H2 have distinct substrate preferences and distinct but overlapping functions in the cell. In prokaryotes and lower eukaryotes, neither enzyme is essential, whereas both are believed to be essential in higher eukaryotes.

Ribonuclease H1 enzymes require at least four ribonucleotide containing base pairs in a substrate and cannot remove a single ribonucleotide from a strand that is otherwise composed of deoxyribonucleotides. For this reason, it is considered unlikely that RNase H1 enzymes are involved in the processing of RNA primers from Okazaki fragments during DNA replication. In many eukaryotes, including mammals, RNase H1 genes include a mitochondrial targeting sequence, leading to expression of isoforms with and without the MTS present. As a result, RNase H1 is localized to both mitochondria and the nucleus. In knockout mouse models, RNase H1 null mutants are lethal during embryogenesis due to defects in replicating mitochondrial DNA. The defects in mitochondrial DNA replication induced by loss of RNase H1 are likely due to defects in R loop processing. The B subunit mediates protein protein interactions between the H2 complex and PCNA, which localizes H2 to replication foci. Although both H1 and H2 are present in the mammalian cell nucleus, H2 is the dominant source of RNase H activity there and is important for maintaining genome stability. Unlike HI and HII, which are both widely distributed among prokaryotes, HIII is found in only a few organisms with a scattered taxonomic distribution; it is somewhat more common in archaea and is rarely or never found in the same prokaryotic genome as HI.

The active site of nearly all RNases H contains four negatively charged amino acid residues, known as the DEDD motif; often a histidine e. g. in HIV 1, human or E. coli is also present. Based on experimental evidence and computer simulations the enzyme activates a water molecule bound to one of the metal ions with the conserved histidine. The transition state is associative in nature and forms an intermediate with protonated phosphate and deprotonated alkoxide leaving group.

In small studies, mutations in human RNase H1 have been associated with chronic progressive external ophthalmoplegia, a common feature of mitochondrial disease. The symptoms of AGS closely resemble those of congenital viral infection and are associated with inappropriate upregulation of type I interferon. AGS can also be caused by mutations in other genes: TREX1, SAMHD1, ADAR, and MDA5/IFIH1, all of which are involved in nucleic acid processing. Characterization of mutational distribution in an AGS patient population found 5% of all AGS mutations in RNASEH2A, 36% in 2B, and 12% in 2C.]] Two groups of viruses use reverse transcription as part of their life cycles: retroviruses, which encode their genomes in single stranded RNA and replicate through a double stranded DNA intermediate; and dsDNA RT viruses, which replicate their double stranded DNA genomes through an RNA "pregenome" intermediate. Pathogenic examples include human immunodeficiency virus and hepatitis B virus, respectively. Both encode large multifunctional reverse transcriptase (RT) proteins containing RNase H domains. Retroviral RT proteins from HIV 1 and murine leukemia virus are the best studied members of the family. Retroviral RT is responsible for converting the virus' single stranded RNA genome into double stranded DNA. This process requires three steps: first, RNA dependent DNA polymerase activity produces minus strand DNA from the plus strand RNA template, generating an RNA:DNA hybrid intermediate; second, the RNA strand is destroyed; and third, DNA dependent DNA polymerase activity synthesizes plus strand DNA, generating double stranded DNA as the final product. The second step of this process is carried out by an RNase H domain located at the C terminus of the RT protein. RNase H performs three types of cleaving actions: non specific degradation of the plus strand RNA genome, specific removal of the minus strand tRNA primer, and removal of the plus strand purine rich polypurine tract (PPT) primer. RNase H plays a role in the priming of the plus strand, but not in the conventional method of synthesizing a new primer sequence. Rather RNase H creates a "primer" from the PPT that is resistant to RNase H cleavage. By removing all bases but the PPT, the PPT is used as a marker for the end of the U3 region of its long terminal repeat. Reverse transcriptase inhibitors that specifically inhibit the polymerase function of RT are in widespread clinical use, but not inhibitors of the RNase H function; it is the only enzymatic function encoded by HIV that is not yet targeted by drugs in clinical use.

RNases H are widely distributed and occur in all domains of life. The family belongs to a larger superfamily of nuclease enzymes In prokaryotic genomes, multiple RNase H genes are often present, but there is little correlation between occurrence of HI, HII, and HIII genes and overall phylogenetic relationships, suggesting that horizontal gene transfer may have played a role in establishing the distribution of these enzymes. RNase HI and HIII rarely or never appear in the same prokaryotic genome. When an organism's genome contains more than one RNase H gene, they sometimes have significant differences in activity level. These observations have been suggested to reflect an evolutionary pattern that minimizes functional redundancy among RNase H genes. The evolutionary trajectory of RNase H2 in eukaryotes, especially the mechanism by which eukaryotic homologs became obligate heterotrimers, is unclear; the B and C subunits have no apparent homologs in prokaryotes. Highly sensitive techniques such as surface plasmon resonance can be used for detection. RNase HII can be used to degrade the RNA primer component of an Okazaki fragment or to introduce single stranded nicks at positions containing a ribonucleotide. Of note, the ribonuclease inhibitor protein commonly used as a reagent is not effective at inhibiting the activity of either HI or HII. RNase H activity was subsequently discovered in E. coli and in a sample of oncoviruses with RNA genomes during early studies of viral reverse transcription. It later became clear that calf thymus extract contained more than one protein with RNase H activity and that E. coli contained two RNase H genes. Originally, the enzyme now known as RNase H2 in eukaryotes was designated H1 and vice versa, but the names of the eukaryotic enzymes were switched to match those in E. coli to facilitate comparative analysis, yielding the modern nomenclature in which the prokaryotic enzymes are designated with Roman numerals and the eukaryotic enzymes with Arabic numerals. The prokaryotic RNase HIII, reported in 1999, was the last RNase H subtype to be identified. The S. cerevisiae homolog of the E. coli protein (that is, the H2A subunit) was easily identifiable by bioinformatics when the yeast genome was sequenced, but the corresponding protein was found not to have enzymatic activity in isolation. However, the yeast B and C subunits have very low sequence identity to their homologs in other organisms, and the corresponding human proteins were conclusively identified only after mutations in all three were found to cause Aicardi–Goutières syndrome.