Ribonuclease H
Ribonuclease H 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. A third type, closely related to H2, is found only in a few prokaryotes, whereas H1 and H2 occur in all domains of life. Additionally, RNase H1-like retroviral ribonuclease H domains occur in multidomain reverse transcriptase proteins, which are encoded by retroviruses such as HIV and are required for viral replication.
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.
Classification and nomenclature
Ribonuclease H is a family of endonuclease enzymes with a shared substrate specificity for the RNA strand of RNA-DNA duplexes. By definition, RNases H cleave RNA backbone phosphodiester bonds to leave a 3' hydroxyl and a 5' phosphate group. 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. In E. coli and many other prokaryotes, the rnhA gene encodes HI and the rnhB gene encodes HII. A third related class, called HIII, occurs in a few bacteria and archaea; it is closely related to prokaryotic HII enzymes.
Structure
The structure of RNase H commonly consists of a 5-stranded β-sheet surrounded by a distribution of α-helices. 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.RNases H2 are larger than H1 and usually have additional helices. The domain organization of the enzymes varies; some prokaryotic and most eukaryotic members of the H1 group have an additional small domain at the N-terminus known as the "hybrid binding domain", which facilitates binding to RNA:DNA hybrid duplexes and sometimes confers increased processivity. While all members of the H1 group and the prokaryotic members of the H2 group function as monomers, eukaryotic H2 enzymes are obligate heterotrimers. Prokaryotic HIII enzymes are members of the broader H2 group and share most structural features with H2, with the addition of an N-terminal TATA box binding domain. Retroviral RNase H domains occurring in multidomain reverse transcriptase proteins have structures closely resembling the H1 group.
RNases H1 have been extensively studied to explore the relationships between structure and enzymatic activity. They are also used, especially the E. coli homolog, as model systems to study protein folding. 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.
Function
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. Depending on the differences in their amino acid sequences, these RNases H are classified into type 1 and type 2 RNases H. 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. The combined activity of both H1 and H2 enzymes is associated with maintenance of genome stability due to the enzymes' degradation of the RNA component of R-loops.Ribonuclease H1
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. RNase H1 is not essential in unicellular organisms where it has been investigated; in E. coli, RNase H1 knockouts confer a temperature-sensitive phenotype, and in S. cerevisiae, they produce defects in stress response.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.
Ribonuclease H2
In prokaryotes, RNase H2 is enzymatically active as a monomeric protein. In eukaryotes, it is an obligate heterotrimer composed of a catalytic subunit A and structural subunits B and C. While the A subunit is closely homologous to the prokaryotic RNase H2, the B and C subunits have no apparent homologs in prokaryotes and are poorly conserved at the sequence level even among eukaryotes. The B subunit mediates protein-protein interactions between the H2 complex and PCNA, which localizes H2 to replication foci.Both prokaryotic and eukaryotic H2 enzymes can cleave single ribonucleotides in a strand. however, they have slightly different cleavage patterns and substrate preferences: prokaryotic enzymes have lower processivity and hydrolyze successive ribonucleotides more efficiently than ribonucleotides with a 5' deoxyribonucleotide, while eukaryotic enzymes are more processive and hydrolyze both types of substrate with similar efficiency. The substrate specificity of RNase H2 gives it a role in ribonucleotide excision repair, removing misincorporated ribonucleotides from DNA, in addition to R-loop processing. 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.
Some prokaryotes possess an additional H2-type gene designated RNase HIII in the Roman-numeral nomenclature used for the prokaryotic genes. HIII proteins are more closely related to the H2 group by sequence identity and structural similarity, but have substrate preferences that more closely resemble H1. 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.
Mechanism
The active site of nearly all RNases H contains four negatively charged amino acid residues, known as the DEDD motif; often a histidine is also present.The charged residues bind two metal ions that are required for catalysis; under physiological conditions these are magnesium ions, but manganese also usually supports enzymatic activity, while calcium or high concentration of Mg2+ inhibits activity.
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. The leaving group is protonated via the glutamate which has an elevated pKa and is likely to be protonated.
The mechanism is similar to RNase T and the RuvC subunit in the Cas9 enzyme which both also use a histidine and a two-metal ion mechanism.
The mechanism of the release of the cleaved product is still unresolved. Experimental evidence from time-resolved crystallography and similar nucleases points to a role of a third ion in the reaction recruited to the active site.
In human biology
The human genome contains four genes encoding RNase H:- RNASEH1, an example of the H1 subtype
- RNASEH2A, the catalytic subunit of the trimeric H2 complex
- RNASEH2B, a structural subunit of the trimeric H2 complex
- RNASEH2C, a structural subunit of the trimeric H2 complex