Ribonuclease H (abbreviated RNase H or RNH) is a family of non-
sequence
In mathematics, a sequence is an enumerated collection of objects in which repetitions are allowed and order matters. Like a set, it contains members (also called ''elements'', or ''terms''). The number of elements (possibly infinite) is cal ...
-specific
endonuclease
In molecular biology, endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain (namely DNA or RNA). Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (with regard to sequence), while man ...
enzyme
An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
s that
catalyze the cleavage of
RNA
Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...
in an RNA/
DNA
Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
substrate
Substrate may refer to:
Physical layers
*Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached
** Substrate (aquatic environment), the earthy material that exi ...
via a
hydrolytic
Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution, elimination, and solvation reactions in which water is the nucleophile.
Biological hydrolysis ...
mechanism
Mechanism may refer to:
*Mechanism (economics), a set of rules for a game designed to achieve a certain outcome
**Mechanism design, the study of such mechanisms
*Mechanism (engineering), rigid bodies connected by joints in order to accomplish a ...
. Members of the RNase H family can be found in nearly all organisms, from
bacteria
Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...
to
archaea
Archaea ( ) is a Domain (biology), domain of organisms. Traditionally, Archaea only included its Prokaryote, prokaryotic members, but this has since been found to be paraphyletic, as eukaryotes are known to have evolved from archaea. Even thou ...
to
eukaryote
The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...
s.
The family is divided into evolutionarily related groups with slightly different
substrate
Substrate may refer to:
Physical layers
*Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached
** Substrate (aquatic environment), the earthy material that exi ...
preferences, broadly designated ribonuclease H1 and H2.
The
human genome
The human genome is a complete set of nucleic acid sequences for humans, encoded as the DNA within each of the 23 distinct chromosomes in the cell nucleus. A small DNA molecule is found within individual Mitochondrial DNA, mitochondria. These ar ...
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
A rare disease is any disease that affects a small percentage of the population. In some parts of the world, the term orphan disease describes a rare disease whose rarity results in little or no funding or research for treatments, without financi ...
known as
Aicardi–Goutières syndrome.
A third type, closely related to H2, is found only in a few
prokaryote
A prokaryote (; less commonly spelled procaryote) is a unicellular organism, single-celled organism whose cell (biology), cell lacks a cell nucleus, nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Ancient Gree ...
s,
whereas H1 and H2 occur in all
domains of life.
Additionally, RNase H1-like
retroviral ribonuclease H domains occur in multidomain
reverse transcriptase
A reverse transcriptase (RT) is an enzyme used to convert RNA genome to DNA, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, by retrotransposon mobi ...
proteins, which are encoded by
retrovirus
A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. After invading a host cell's cytoplasm, the virus uses its own reverse transcriptase e ...
es such as
HIV and are required for viral replication.
In eukaryotes, ribonuclease H1 is involved in
DNA replication
In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all life, living organisms, acting as the most essential part of heredity, biolog ...
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
In molecular biology, endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain (namely DNA or RNA). Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (with regard to sequence), while man ...
enzyme
An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
s with a shared
substrate
Substrate may refer to:
Physical layers
*Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached
** Substrate (aquatic environment), the earthy material that exi ...
specificity for the RNA strand of
RNA
Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...
-
DNA
Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
duplexes. By definition, RNases H cleave RNA backbone
phosphodiester bonds to leave a
3' hydroxyl
In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydroxy ...
and a
5' phosphate
Phosphates are the naturally occurring form of the element phosphorus.
In chemistry, a phosphate is an anion, salt, functional group or ester derived from a phosphoric acid. It most commonly means orthophosphate, a derivative of orthop ...
group.
RNases H have been proposed as members of an evolutionarily related superfamily encompassing other
nuclease
In biochemistry, a nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds that link nucleotides together to form nucleic acids. Nucleases variously affect single and ...
s and nucleic acid processing enzymes such as
retroviral integrases, DNA
transposase
A transposase is any of a class of enzymes capable of binding to the end of a transposon and catalysing its movement to another part of a genome, typically by a cut-and-paste mechanism or a replicative mechanism, in a process known as transpositio ...
s,
Holliday junction resolvases,
Piwi and
Argonaute proteins, various
exonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is th ...
s, 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
eukaryote
The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...
s and Roman numeral designations in
prokaryote
A prokaryote (; less commonly spelled procaryote) is a unicellular organism, single-celled organism whose cell (biology), cell lacks a cell nucleus, nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Ancient Gree ...
s. Thus the ''
Escherichia coli
''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Escherichia'' that is commonly fo ...
'' RNase HI is a homolog of the ''
Homo sapiens
Humans (''Homo sapiens'') or modern humans are the most common and widespread species of primate, and the last surviving species of the genus ''Homo''. They are Hominidae, great apes characterized by their Prehistory of nakedness and clothing ...
'' 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
Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...
and
archaea
Archaea ( ) is a Domain (biology), domain of organisms. Traditionally, Archaea only included its Prokaryote, prokaryotic members, but this has since been found to be paraphyletic, as eukaryotes are known to have evolved from archaea. Even thou ...
; it is closely related to prokaryotic HII enzymes.
Structure

The
structure
A structure is an arrangement and organization of interrelated elements in a material object or system, or the object or system so organized. Material structures include man-made objects such as buildings and machines and natural objects such as ...
of RNase H commonly consists of a 5-stranded
β-sheet
The beta sheet (β-sheet, also β-pleated sheet) is a common structural motif, motif of the regular protein secondary structure. Beta sheets consist of beta strands (β-strands) connected laterally by at least two or three backbone chain, backbon ...
surrounded by a distribution of
α-helices.
All RNases H have an
active site
In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding s ...
centered on a conserved
sequence motif
In biology, a sequence motif is a nucleotide or amino-acid sequence pattern that is widespread and usually assumed to be related to biological function of the macromolecule. For example, an ''N''-glycosylation site motif can be defined as ''A ...
composed of
aspartate
Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. The L-isomer of aspartic acid is one of the 22 proteinogenic amino acids, i.e., the building blocks of protein ...
and
glutamate
Glutamic acid (symbol Glu or E; known as glutamate in its anionic form) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a Essential amino acid, non-essential nutrient for humans, meaning that ...
residues, often referred to as the DEDD motif. These residues interact with catalytically required
magnesium
Magnesium is a chemical element; it has Symbol (chemistry), symbol Mg and atomic number 12. It is a shiny gray metal having a low density, low melting point and high chemical reactivity. Like the other alkaline earth metals (group 2 ...
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
The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...
known as the "hybrid binding domain", which facilitates binding to RNA:DNA hybrid duplexes and sometimes confers increased processivity
In molecular biology and biochemistry, processivity is an enzyme's ability to catalyze "consecutive reactions without releasing its substrate".
For example, processivity is the average number of nucleotides added by a polymerase enzyme, such as ...
. 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
A reverse transcriptase (RT) is an enzyme used to convert RNA genome to DNA, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, by retrotransposon mobi ...
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
Protein folding is the physical process by which a protein, after Protein biosynthesis, synthesis by a ribosome as a linear chain of Amino acid, amino acids, changes from an unstable random coil into a more ordered protein tertiary structure, t ...
. 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
Basic or BASIC may refer to:
Science and technology
* BASIC, a computer programming language
* Basic (chemistry), having the properties of a base
* Basic access authentication, in HTTP
Entertainment
* Basic (film), ''Basic'' (film), a 2003 film
...
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
Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...
in a double-stranded RNA:DNA hybrid, leaving a 3' hydroxyl
In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydroxy ...
and a 5' phosphate
Phosphates are the naturally occurring form of the element phosphorus.
In chemistry, a phosphate is an anion, salt, functional group or ester derived from a phosphoric acid. It most commonly means orthophosphate, a derivative of orthop ...
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
Substrate may refer to:
Physical layers
*Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached
** Substrate (aquatic environment), the earthy material that exi ...
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
A genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding genes, other functional regions of the genome such as ...
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 pair
A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
s 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 fragment
Okazaki fragments are short sequences of DNA nucleotides (approximately 150 to 200 base pairs long in eukaryotes) which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA ...
s during DNA replication
In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all life, living organisms, acting as the most essential part of heredity, biolog ...
. RNase H1 is not essential in unicellular organisms where it has been investigated; in '' E. coli'', RNase H1 knockout
A knockout (abbreviated to KO or K.O.) is a fight-ending, winning criterion in several full-contact combat sports, such as boxing, kickboxing, Muay Thai, mixed martial arts, karate, some forms of taekwondo and other sports involving striking, ...
s confer a temperature-sensitive phenotype, and in '' S. cerevisiae'', they produce defects in stress response.
In many eukaryotes, including mammal
A mammal () is a vertebrate animal of the Class (biology), class Mammalia (). Mammals are characterised by the presence of milk-producing mammary glands for feeding their young, a broad neocortex region of the brain, fur or hair, and three ...
s, RNase H1 genes include a mitochondrial targeting sequence, leading to expression of isoforms
A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene and are the result of genetic differences. While many perform the same or similar biological roles, some isoforms have uniqu ...
with and without the MTS present. As a result, RNase H1 is localized to both mitochondria
A mitochondrion () is an organelle found in the cells of most eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is us ...
and the nucleus. In knockout mouse
A knockout mouse, or knock-out mouse, is a genetically modified mouse (''Mus musculus'') in which researchers have inactivated, or " knocked out", an existing gene by replacing it or disrupting it with an artificial piece of DNA. They are importan ...
models, RNase H1-null mutants are lethal during embryogenesis
An embryo ( ) is the initial stage of development for a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male ...
due to defects in replicating mitochondrial DNA
Mitochondrial DNA (mtDNA and mDNA) is the DNA located in the mitochondrion, mitochondria organelles in a eukaryotic cell that converts chemical energy from food into adenosine triphosphate (ATP). Mitochondrial DNA is a small portion of the D ...
. 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
In mathematics, a sequence is an enumerated collection of objects in which repetitions are allowed and order matters. Like a set, it contains members (also called ''elements'', or ''terms''). The number of elements (possibly infinite) is cal ...
level even among eukaryotes. The B subunit mediates protein-protein interactions between the H2 complex and PCNA
Proliferating cell nuclear antigen (PCNA) is a DNA clamp that acts as a processivity factor for DNA polymerase delta, DNA polymerase δ in eukaryotic cell (biology), cells and is essential for replication. PCNA is a homotrimer and achieves its ...
, 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
In molecular biology and biochemistry, processivity is an enzyme's ability to catalyze "consecutive reactions without releasing its substrate".
For example, processivity is the average number of nucleotides added by a polymerase enzyme, such as ...
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
The cell nucleus (; : nuclei) is a membrane-bound organelle found in eukaryote, eukaryotic cell (biology), cells. Eukaryotic cells usually have a single nucleus, but a few cell types, such as mammalian red blood cells, have #Anucleated_cells, ...
, 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
Archaea ( ) is a Domain (biology), domain of organisms. Traditionally, Archaea only included its Prokaryote, prokaryotic members, but this has since been found to be paraphyletic, as eukaryotes are known to have evolved from archaea. Even thou ...
and is rarely or never found in the same prokaryotic genome as HI.
Mechanism
The active site
In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding s ...
of nearly all RNases H contains four negatively charged amino acid residues, known as the DEDD motif; often a histidine
Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an Amine, α-amino group (which is in the protonated –NH3+ form under Physiological condition, biological conditions), a carboxylic ...
e.g. in HIV-1, human or E. coli is also present.
The charged residues bind two metal ions that are required for catalysis; under physiological conditions these are magnesium
Magnesium is a chemical element; it has Symbol (chemistry), symbol Mg and atomic number 12. It is a shiny gray metal having a low density, low melting point and high chemical reactivity. Like the other alkaline earth metals (group 2 ...
ions, but manganese
Manganese is a chemical element; it has Symbol (chemistry), symbol Mn and atomic number 25. It is a hard, brittle, silvery metal, often found in minerals in combination with iron. Manganese was first isolated in the 1770s. It is a transition m ...
also usually supports enzymatic activity, while calcium
Calcium is a chemical element; it has symbol Ca and atomic number 20. As an alkaline earth metal, calcium is a reactive metal that forms a dark oxide-nitride layer when exposed to air. Its physical and chemical properties are most similar to it ...
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
In chemistry, the transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest potential energy along this reaction coordinate. It is often marked w ...
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
Ribonuclease T (''RNase T'', ''exonuclease T'', ''exo T'') is a ribonuclease enzyme involved in the maturation of transfer RNA and ribosomal RNA in bacteria, as well as in DNA repair pathways. It is a member of the DnaQ family of exonucleases ...
and the RuvC subunit in the Cas9
Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 dalton (unit), kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utili ...
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
The human genome is a complete set of nucleic acid sequences for humans, encoded as the DNA within each of the 23 distinct chromosomes in the cell nucleus. A small DNA molecule is found within individual Mitochondrial DNA, mitochondria. These ar ...
contains four genes encoding RNase H:
* RNASEH1, an example of the H1 (monomeric) 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
In addition, genetic material of retroviral origin appears frequently in the genome, reflecting integration of the genomes of human endogenous retrovirus
Endogenous retroviruses (ERVs) are endogenous viral elements in the genome that closely resemble and can be derived from retroviruses. They are abundant in the genomes of Gnathostomata, jawed vertebrates, and they comprise up to 5–8% of the hu ...
es. Such integration events result in the presence of genes encoding retroviral reverse transcriptase
A reverse transcriptase (RT) is an enzyme used to convert RNA genome to DNA, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, by retrotransposon mobi ...
, which includes an RNase H domain. An example is ERVK6. Long terminal repeat
A long terminal repeat (LTR) is a pair of identical sequences of DNA, several hundred base pairs long, which occur in eukaryotic genomes on either end of a series of genes or pseudogenes that form a retrotransposon or an endogenous retrovirus o ...
(LTR) and non-long terminal repeat (non-LTR) retrotransposon
Retrotransposons (also called Class I transposable elements) are mobile elements which move in the host genome by converting their transcribed RNA into DNA through reverse transcription. Thus, they differ from Class II transposable elements, or ...
s are also common in the genome and often include their own RNase H domains, with a complex evolutionary history.
Role in disease
In small studies, mutations in human RNase H1 have been associated with chronic progressive external ophthalmoplegia, a common feature of mitochondrial disease.
Mutations in any of the three RNase H2 subunits are well-established as causes of a rare genetic disorder
A genetic disorder is a health problem caused by one or more abnormalities in the genome. It can be caused by a mutation in a single gene (monogenic) or multiple genes (polygenic) or by a chromosome abnormality. Although polygenic disorders ...
known as Aicardi–Goutières syndrome (AGS), which manifests as neurological
Neurology (from , "string, nerve" and the suffix -logia, "study of") is the branch of medicine dealing with the diagnosis and treatment of all categories of conditions and disease involving the nervous system, which comprises the brain, the s ...
and dermatological symptoms at an early age. 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
Adar (Hebrew: , ; from Akkadian ''adaru'') is the sixth month of the civil year and the twelfth month of the religious year on the Hebrew calendar, roughly corresponding to the month of March in the Gregorian calendar. It is a month of 29 days. ...
, 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. Mutations in 2B have been associated with somewhat milder neurological impairment[ and with an absence of interferon-induced gene upregulation that can be detected in patients with other AGS-associated genotypes.]
In viruses
Two groups of virus
A virus is a submicroscopic infectious agent that replicates only inside the living Cell (biology), cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Viruses are ...
es use reverse transcription
A reverse transcriptase (RT) is an enzyme used to convert RNA genome to DNA, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B virus, hepatitis B to replicate their genomes, by retrot ...
as part of their life cycles: retrovirus
A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. After invading a host cell's cytoplasm, the virus uses its own reverse transcriptase e ...
es, 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. Pathogen
In biology, a pathogen (, "suffering", "passion" and , "producer of"), in the oldest and broadest sense, is any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a Germ theory of d ...
ic examples include human immunodeficiency virus
The human immunodeficiency viruses (HIV) are two species of ''Lentivirus'' (a subgroup of retrovirus) that infect humans. Over time, they cause AIDS, acquired immunodeficiency syndrome (AIDS), a condition in which progressive failure of th ...
and hepatitis B virus
Hepatitis B virus (HBV) is a partially double-stranded DNA virus, a species of the genus '' Orthohepadnavirus'' and a member of the '' Hepadnaviridae'' family of viruses. This virus causes the disease hepatitis B.
Classification
Hepatitis B ...
, respectively. Both encode large multifunctional reverse transcriptase
A reverse transcriptase (RT) is an enzyme used to convert RNA genome to DNA, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, by retrotransposon mobi ...
(RT) proteins containing RNase H domains.
Retroviral RT proteins from HIV-1
The subtypes of HIV include two main subtypes, known as HIV type 1 (HIV-1) and HIV type 2 (HIV-2). These subtypes have distinct genetic differences and are associated with different epidemiological patterns and clinical characteristics.
HIV-1 e ...
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
The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein
Proteins are large biomolecules and macromolecules that comp ...
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
Transfer ribonucleic acid (tRNA), formerly referred to as soluble ribonucleic acid (sRNA), is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length (in eukaryotes). In a cell, it provides the physical link between the gene ...
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
A long terminal repeat (LTR) is a pair of identical sequences of DNA, several hundred base pairs long, which occur in eukaryotic genomes on either end of a series of genes or pseudogenes that form a retrotransposon or an endogenous retrovirus o ...
.
Because RNase H activity is required for viral proliferation, this domain has been considered a drug target
A biological target is anything within a living organism to which some other entity (like an endogenous ligand or a drug) is directed and/or binds, resulting in a change in its behavior or function. Examples of common classes of biological targets ...
for the development of antiretroviral
The management of HIV/AIDS normally includes the use of multiple antiretroviral drugs as a strategy to control HIV infection. There are several classes of antiretroviral agents that act on different stages of the HIV life-cycle. The use of mu ...
drugs used in the treatment of HIV/AIDS
The HIV, human immunodeficiency virus (HIV) is a retrovirus that attacks the immune system. Without treatment, it can lead to a spectrum of conditions including acquired immunodeficiency syndrome (AIDS). It is a Preventive healthcare, pr ...
and other conditions caused by retroviruses. Inhibitors of retroviral RNase H of several different chemotype A chemotype (sometimes chemovar) is a chemically distinct entity in a plant or microorganism, with differences in the composition of the secondary metabolites. Minor genetic and epigenetic changes with little or no effect on morphology or anatomy ...
s have been identified, many of which have a mechanism of action
In pharmacology, the term mechanism of action (MOA) refers to the specific biochemical Drug interaction, interaction through which a Medication, drug substance produces its pharmacological effect. A mechanism of action usually includes mention o ...
based on chelation
Chelation () is a type of bonding of ions and their molecules to metal ions. It involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central metal atom. These l ...
of the active-site cations. 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.
Evolution
RNases H are widely distributed and occur in all domains of life. The family belongs to a larger superfamily of nuclease
In biochemistry, a nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds that link nucleotides together to form nucleic acids. Nucleases variously affect single and ...
enzymes and is considered to be evolutionarily ancient. 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
Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the movement of genetic material between organisms other than by the ("vertical") transmission of DNA from parent to offspring (reproduction). HGT is an important factor in the e ...
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. RNase HIII, which is unique to prokaryotes, has a scattered taxonomic distribution and is found in both bacteria
Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...
and archaea
Archaea ( ) is a Domain (biology), domain of organisms. Traditionally, Archaea only included its Prokaryote, prokaryotic members, but this has since been found to be paraphyletic, as eukaryotes are known to have evolved from archaea. Even thou ...
; it is believed to have diverged from HII fairly early.
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.
Applications
Because RNase H specifically degrades only the RNA in double-stranded RNA:DNA hybrids, it is commonly used as a laboratory reagent in molecular biology
Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...
. Purified preparations of ''E. coli'' RNase HI and HII are commercially available. RNase HI is often used to destroy the RNA template after first-strand complementary DNA
In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engin ...
(cDNA) synthesis by reverse transcription
A reverse transcriptase (RT) is an enzyme used to convert RNA genome to DNA, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B virus, hepatitis B to replicate their genomes, by retrot ...
. It can also be used to cleave specific RNA sequences in the presence of short complementary segments of DNA. Highly sensitive techniques such as surface plasmon resonance
Surface plasmon resonance (SPR) is a phenomenon that occurs where electrons in a thin metal sheet become excited by light that is directed to the sheet with a particular angle of incidence (optics), angle of incidence, and then travel parallel to ...
can be used for detection. RNase HII can be used to degrade the RNA primer component of an Okazaki fragment
Okazaki fragments are short sequences of DNA nucleotides (approximately 150 to 200 base pairs long in eukaryotes) which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA ...
or to introduce single-stranded nicks at positions containing a ribonucleotide. A variant of hot start PCR, known as RNase H-dependent PCR or rhPCR, has been described using a thermostable RNase HII from the hyperthermophilic archaeon '' Pyrococcus abyssi''. Of note, the ribonuclease inhibitor
Ribonuclease inhibitor (RI) is a large (~450 residues, ~49 kDa), acidic (pI ~4.7), leucine-rich repeat protein that forms extremely tight complexes with certain ribonucleases. It is a major cellular protein, comprising ~0.1% of all cellular prote ...
protein commonly used as a reagent is not effective at inhibiting the activity of either HI or HII.
History
Ribonucleases H were first discovered in the laboratory of Peter Hausen when researchers found RNA:DNA hybrid endonuclease
In molecular biology, endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain (namely DNA or RNA). Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (with regard to sequence), while man ...
activity in calf thymus
The thymus (: thymuses or thymi) is a specialized primary lymphoid organ of the immune system. Within the thymus, T cells mature. T cells are critical to the adaptive immune system, where the body adapts to specific foreign invaders. The thymus ...
in 1969 and gave it the name "ribonuclease ''H''" to designate its ''hybrid'' specificity. RNase H activity was subsequently discovered in '' E. coli'' and in a sample of oncovirus
An oncovirus or oncogenic virus is a virus that can cause cancer. This term originated from studies of acutely transforming retroviruses in the 1950–60s, when the term ''oncornaviruses'' was used to denote their RNA virus origin. With the let ...
es with RNA genomes during early studies of viral reverse transcription
A reverse transcriptase (RT) is an enzyme used to convert RNA genome to DNA, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B virus, hepatitis B to replicate their genomes, by retrot ...
. 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.
Characterizing eukaryotic RNase H2 was historically a challenge, in part due to its low abundance. Careful efforts at purification of the enzyme suggested that, unlike the ''E. coli'' RNase H2, the eukaryotic enzyme had multiple subunits. The '' S. cerevisiae'' homolog of the ''E. coli'' protein (that is, the H2A subunit) was easily identifiable by bioinformatics
Bioinformatics () is an interdisciplinary field of science that develops methods and Bioinformatics software, software tools for understanding biological data, especially when the data sets are large and complex. Bioinformatics uses biology, ...
when the yeast genome
A genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding genes, other functional regions of the genome such as ...
was sequenced, but the corresponding protein was found not to have enzymatic activity in isolation. Eventually, the yeast B and C subunits were isolated by co-purification and found to be required for enzymatic activity. 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.
References
External links
GeneReviews/NCBI/NIH/UW entry on Aicardi-Goutières Syndrome
*
{{DEFAULTSORT:Rnase H
Ribonucleases
EC 3.1.26