DNA-3-methyladenine Glycosylase
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DNA-3-methyladenine glycosylase also known as 3-alkyladenine DNA glycosylase (AAG) or N-methylpurine DNA glycosylase (MPG) is an
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
that in humans is encoded by the ''MPG''
gene In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
. Alkyladenine DNA glycosylase is a specific type of
DNA glycosylase DNA glycosylases are a family of enzymes involved in base excision repair, classified under EC number EC 3.2.2. Base excision repair is the mechanism by which damaged bases in DNA are removed and replaced. DNA glycosylases catalyze the first st ...
. This subfamily of monofunctional glycosylases is involved in the recognition of a variety of base lesions, including alkylated and deaminated purines, and initiating their repair via the
base excision repair Base excision repair (BER) is a cellular mechanism, studied in the fields of biochemistry and genetics, that repairs damaged DNA throughout the cell cycle. It is responsible primarily for removing small, non-helix-distorting base lesions from t ...
pathway. To date, the human AAG (hAAG) is the only glycosylase identified that excises alkylation-damaged purine bases in human cells.


Function

DNA bases are subject to a large number of anomalies: spontaneous
alkylation Alkylation is the transfer of an alkyl group from one molecule to another. The alkyl group may be transferred as an alkyl carbocation, a free radical, a carbanion, or a carbene (or their equivalents). Alkylating agents are reagents for effecting ...
or oxidative
deamination Deamination is the removal of an amino group from a molecule. Enzymes that catalyse this reaction are called deaminases. In the human body, deamination takes place primarily in the liver, however it can also occur in the kidney. In situations of e ...
. It is estimated that 104 mutations appear in a typical human cell per day. Albeit it seems to be an insignificant amount considering the extension of the DNA (1010 nucleotides), these
mutation In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mi ...
s lead to changes in the structure and coding potential of the DNA, affecting processes of replication and
transcription Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including: Genetics * Transcription (biology), the copying of DNA into RNA, the fir ...
. 3-Methyladenine DNA glycosilases are able to initiate the
base excision repair Base excision repair (BER) is a cellular mechanism, studied in the fields of biochemistry and genetics, that repairs damaged DNA throughout the cell cycle. It is responsible primarily for removing small, non-helix-distorting base lesions from t ...
(BER) of a wide range of substrate bases that, due to their chemical reactivity, suffer inevitable modifications resulting in different biological outcomes. DNA repair mechanisms take on a vital role in maintaining the genomic integrity of cells from different organisms, in particular 3-Methyladenine DNA glycosylases are found in
bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
,
yeast Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitut ...
,
plant Plants are predominantly photosynthetic eukaryotes of the kingdom Plantae. Historically, the plant kingdom encompassed all living things that were not animals, and included algae and fungi; however, all current definitions of Plantae exclud ...
s,
rodent Rodents (from Latin , 'to gnaw') are mammals of the order Rodentia (), which are characterized by a single pair of continuously growing incisors in each of the upper and lower jaws. About 40% of all mammal species are rodents. They are na ...
s and
human Humans (''Homo sapiens'') are the most abundant and widespread species of primate, characterized by bipedalism and exceptional cognitive skills due to a large and complex brain. This has enabled the development of advanced tools, culture, ...
s. Therefore, there are different subfamilies of this enzyme, such as the Human Alkyladenine DNA Glycosylase (hAAG), that act on other damaged DNA bases apart from 3-MeA.


Alkylation repairing activity

In cells, AAG is the
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
responsible for recognition and initiation of the repair, via catalysing the hydrolysis of the N-glycosidic bond to release the alkylation-damaged purine bases. Specifically, hAAG is able to efficiently identify and excise 3-methyladenine, 7-methyladenine,
7-methylguanine 7-Methylguanine is a modified purine nucleobase. It is a methylated version of guanine. The 7-methylguanine nucleoside is called 7-methylguanosine 7-Methylguanosine (m7G) is a modified purine nucleoside. It is a methylated version of guano ...
, 1N-ethenoadenine and
hypoxanthine Hypoxanthine is a naturally occurring purine derivative. It is occasionally found as a constituent of nucleic acids, where it is present in the anticodon of tRNA in the form of its nucleoside inosine. It has a tautomer known as 6-hydroxypurine. Hyp ...
.


ODG activity

Oxanine DNA Glycolase (ODG) activity is the capability of some DNA glycosylases of repairing oxanines (Oxa), a deaminated base lesion in which the N1-nitrogen is replaced by oxygen. Among the known human DNA glycosylases tested, the human alkyladenine DNA glycosylase (AAG) also shows ODG activity. Contrary to the alkylation repairing activity, which is only able to act against purine bases, the hAAG is able to excise Oxa from all of four Oxa-containing double stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, showing no particular preference by any of the bases. In addition hAAG is capable of removing Oxa from single-stranded Oxa- containing DNA. This occurs because the ODG activity of the hAAG does not require a complementary strand.


Structure

Alkyladenine DNA glycosylase is a monomeric
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
compounded by 298
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
s, with a formula weight of 33kDa. Its canonical primary structure consists of the following sequence. However, also other functional isoforms have been found.


Isoform 2

The sequence of this isoform differs from the canonical sequence as follows: Aminoacids 1-12: MVTPALQMKKPK → MPARSGA Aminoacids 195-196: QL →HV


Isoform 3

The sequence of this isoform differs from the canonical sequence in a similar way as the isoform 2: Aminoacids 1-12: MVTPALQMKKPK → MPARSGA


Isoform 4

The sequence of this isoform misses the aminoacids 1-17. It folds into a single domain of mixed α/β structure with seven
α helices The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ear ...
and eight β strands. The core of the protein consists of a curved, antiparallel β sheet with a protruding β hairpin (β3β4) that inserts into the minor groove of the bound DNA. A series of α helices and connecting loops form the remainder of the DNA binding interface. It lacks the helix-hairpin-helix motif associated with other base excision-repair proteins and, in fact, it does not resemble any other model in the
Protein Data Bank The Protein Data Bank (PDB) is a database for the three-dimensional structural data of large biological molecules, such as proteins and nucleic acids. The data, typically obtained by X-ray crystallography, NMR spectroscopy, or, increasingly, cry ...
.


Mechanism


Substrate recognition

Alkyladenine DNA glycosylase is part of the family of
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
s that follow the
BER ''Ziziphus mauritiana'', also known as Indian jujube, Indian plum, Chinese date, Chinese apple, ber, and dunks is a tropical fruit tree species belonging to the family Rhamnaceae. It is often confused with the closely related jujube, Chinese j ...
, acting on specific substrates according to BER steps. The process of recognition of damaged bases involves initial non-specific binding followed by diffusion along the DNA. Formed the AAG-DNA complex, a redundant process of search occurs because of the long lifetime of this complex, while hAAG search many adjacent sites in a DNA molecule in a single binding. This provides ample opportunity to recognize and excise lesions that minimally perturb the structure of the DNA. Due to its broad specificity, the hAAG performs the substrate selection through a combination of selectivity filters. * The first selectivity filter occurs at the nucleotide flipping step of unusable base pairs that present lesions. * The second selectivity filter is constituted by the catalytic mechanism which ensures that only purine bases are excised, even though smaller pyrimidines can fit in the hAAG’s
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 (binding site) a ...
. The active site pocket it’s designed to accommodate a structurally diverse set of modified purines so it would be difficult to sterically exclude the smaller pyrimidine bases from binding. However, thanks to the different shape and chemical properties of a bound pyrimidine and a purine substrate, the acid-catalyzed reacts only with the pyrimidine preventing it from binding with the hAAG. * The third selectivity filter consist of unfavorable steric clashes that allow a preferential recognition of purine lesions lacking exocyclic amino groups of
guanine Guanine () ( symbol G or Gua) is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine (uracil in RNA). In DNA, guanine is paired with cytosine. The guanine nucleoside is called ...
and adenine.


Nucleotide flipping and fixation

Its structure contains an antiparallel β sheet with protruding β hairpin (β3β4) that inserts into the minor groove of the bound DNA. This group is unique for the human cells and displaces the selected nucleotide targeted for base excision by flipping it. The nucleotide is secured into the enzyme binding pocket where the active site is found, and is fixed by the amino acids Arg182, Glu125 and Ser262. Also other bonds are formed to bordering nucleotides to stabilize the structure. The groove in the double helix of DNA left by the flipped-out abasic nucleotide is filled with the lateral chain of the amino acid Tyr162, making no specific contacts with the surrounding bases.


Nucleotide release

Activated by nearby aminoacids, a water molecule attacks the N-Glycosydic bound releasing the alkylated base via a backside displacement mechanism.


Location

Human alkyladenine DNA glycosylase localizes to the
mitochondria A mitochondrion (; ) is an organelle found in the Cell (biology), cells of most Eukaryotes, such as animals, plants and Fungus, fungi. Mitochondria have a double lipid bilayer, membrane structure and use aerobic respiration to generate adenosi ...
,
nucleus Nucleus ( : nuclei) is a Latin word for the seed inside a fruit. It most often refers to: *Atomic nucleus, the very dense central region of an atom *Cell nucleus, a central organelle of a eukaryotic cell, containing most of the cell's DNA Nucle ...
and
cytoplasm In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. The ...
of human cells. Some functionally equivalent enzymes have been found in other species have significantly different structures, such as
DNA-3-methyladenine glycosylase DNA-3-methyladenine glycosylase also known as 3-alkyladenine DNA glycosylase (AAG) or N-methylpurine DNA glycosylase (MPG) is an enzyme that in humans is encoded by the ''MPG'' gene. Alkyladenine DNA glycosylase is a specific type of DNA glycos ...
in E. coli.


Clinical significance

According to the mechanism used by Human Alkyladenine DNA Glycosylase, a defect in the DNA repair pathways leads to
cancer Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
predisposition. HAAG follows the BER steps so that means that an incorrect role of BER genes could contribute to the development of cancer. Concretely, a bad activity of hAAG may be associated with cancer risk in
BRCA1 Breast cancer type 1 susceptibility protein is a protein that in humans is encoded by the ''BRCA1'' () gene. Orthologs are common in other vertebrate species, whereas invertebrate genomes may encode a more distantly related gene. ''BRCA1'' is a h ...
and
BRCA2 ''BRCA2'' and BRCA2 () are a human gene and its protein product, respectively. The official symbol (BRCA2, italic for the gene, nonitalic for the protein) and the official name (originally breast cancer 2; currently BRCA2, DNA repair associated) ...
mutation carriers.


Aging

As noted above, DNA-3-methyladenine glycosylase (also called 3-alklyadeneine DNA glycosylase or AAG) is able to identify and excise a variety of alkylation damaged
purine Purine is a heterocyclic compound, heterocyclic aromatic organic compound that consists of two rings (pyrimidine and imidazole) fused together. It is water-soluble. Purine also gives its name to the wider class of molecules, purines, which includ ...
bases. Such damages to purine bases occur spontaneously in DNA. Double-mutant mice deficient both for AAG and another enzyme that specifically repairs O6MeG damages (
O-6-methylguanine-DNA methyltransferase ''O''6-alkylguanine DNA alkyltransferase (also known as AGT, MGMT or AGAT) is a protein that in humans is encoded by the ''O''6-methylguanine DNA methyltransferase (''MGMT'') gene. O6-methylguanine DNA methyltransferase is crucial for genome stabi ...
) had a shorter lifespan and aged more rapidly than wild type mice. These findings indicate that damaged purine bases contribute to the aging process, consistent with the
DNA damage theory of aging The DNA damage theory of aging proposes that aging is a consequence of unrepaired accumulation of naturally occurring DNA damage. Damage in this context is a DNA alteration that has an abnormal structure. Although both mitochondrial and nuclear ...
.


Model organisms

Model organism A model organism (often shortened to model) is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model organism will provide insight into the workin ...
s have been used in the study of MPG function. A conditional
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 ...
line called ''Mpgtm1a(EUCOMM)Wtsi'' was generated at the
Wellcome Trust Sanger Institute The Wellcome Sanger Institute, previously known as The Sanger Centre and Wellcome Trust Sanger Institute, is a non-profit British genomics and genetics research institute, primarily funded by the Wellcome Trust. It is located on the Wellcome G ...
. Male and female animals underwent a standardized
phenotypic screen In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
to determine the effects of deletion. Additional screens performed: - In-depth immunological phenotyping


See also

*
DNA-3-methyladenine glycosylase II DNA-3-methyladenine glycosylase II () is an enzyme that catalyses the following chemical reaction: : Hydrolysis of alkylated DNA, releasing 3-methyladenine, 3-methylguanine, 7-methylguanine, and 7-methyladenine Involved in the removal of a ...


References


Further reading

* * * * * * * * * * * * * * * * *


External links

* {{Portal bar, Biology, border=no EC 3.2.2