DNA glycosylases are a 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 ...

s involved in

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 ...

, classified under EC number EC 3.2.2.

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 ...

is the mechanism by which damaged bases in DNA are removed and replaced. DNA glycosylases catalyze the first step of this process. They remove the damaged nitrogenous base while leaving the sugar-phosphate backbone intact, creating an apurinic/apyrimidinic site, commonly referred to as an

AP site In biochemistry and molecular genetics, an AP site (apurinic/apyrimidinic site), also known as an abasic site, is a location in DNA (also in RNA but much less likely) that has neither a purine nor a pyrimidine base, either spontaneously or due ...

. This is accomplished by

flipping Flipping is a term used to describe purchasing a revenue-generating asset and quickly reselling (or "flipping") it for profit. Within the real estate industry, the term is used by investors to describe the process of buying, rehabbing, and sel ...

the damaged base out of the double helix followed by cleavage of the N-

glycosidic bond A glycosidic bond or glycosidic linkage is a type of covalent bond that joins a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate. A glycosidic bond is formed between the hemiacetal or hemiketal group ...

. Glycosylases were first discovered in bacteria, and have since been found in all kingdoms of life. In addition to their role in base excision repair, DNA glycosylase enzymes have been implicated in the repression of gene silencing in ''A. thaliana'', ''N. tabacum'' and other plants by active demethylation. 5-methylcytosine residues are excised and replaced with unmethylated cytosines allowing access to the chromatin structure of the enzymes and proteins necessary for transcription and subsequent translation.

Monofunctional vs. bifunctional glycosylases

There are two main classes of glycosylases: monofunctional and bifunctional. Monofunctional glycosylases have only glycosylase activity, whereas bifunctional glycosylases also possess AP lyase activity that permits them to cut the

phosphodiester bond In chemistry, a phosphodiester bond occurs when exactly two of the hydroxyl groups () in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. The "bond" involves this linkage . Discussion of phosphodiesters is d ...

of DNA, creating a single-strand break without the need for an

AP endonuclease Apurinic/apyrimidinic (AP) endonuclease is an enzyme that is involved in the DNA base excision repair pathway (BER). Its main role in the repair of damaged or mismatched nucleotides in DNA is to create a nick in the phosphodiester backbone of t ...

. β-Elimination of an AP site by a glycosylase-lyase yields a 3' α,β-unsaturated aldehyde adjacent to a 5' phosphate, which differs from the AP endonuclease cleavage product. Some glycosylase-lyases can further perform δ-elimination, which converts the 3' aldehyde to a 3' phosphate.

Biochemical mechanism

The first

crystal structure In crystallography, crystal structure is a description of the ordered arrangement of atoms, ions or molecules in a crystalline material. Ordered structures occur from the intrinsic nature of the constituent particles to form symmetric patterns ...

of a DNA glycosylase was obtained for E. coli Nth. This structure revealed that the enzyme flips the damaged base out of the double helix into an active site pocket in order to excise it. Other glycosylases have since been found to follow the same general paradigm, including human UNG pictured below. To cleave the N-glycosidic bond, monofunctional glycosylases use an activated water molecule to attack carbon 1 of the substrate. Bifunctional glycosylases, instead, use an amine residue as a nucleophile to attack the same carbon, going through a

Schiff base In organic chemistry, a Schiff base (named after Hugo Schiff) is a compound with the general structure ( = alkyl or aryl, but not hydrogen). They can be considered a sub-class of imines, being either secondary ketimines or secondary aldimine ...

intermediate.

Types of glycosylases

Crystal structures A crystal or crystalline solid is a solid material whose constituents (such as atoms, molecules, or ions) are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. In addition, macrosc ...

of many glycosylases have been solved. Based on structural similarity, glycosylases are grouped into four superfamilies. The UDG and AAG families contain small, compact glycosylases, whereas the MutM/Fpg and HhH-GPD families comprise larger enzymes with multiple domains. A wide variety of glycosylases have evolved to recognize different damaged bases. The table below summarizes the properties of known glycosylases in commonly studied model organisms. DNA glycosylases can be grouped into the following categories based on their substrate(s):

Uracil DNA glycosylases

In molecular biology, the

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, res ...

family,

Uracil-DNA glycosylase Uracil-DNA glycosylase is also known as UNG or UDG. Its most important function is to prevent mutagenesis by eliminating uracil from DNA molecules by cleaving the N-glycosidic bond and initiating the base-excision repair (BER) pathway. Function ...

(UDG) is an

that reverts mutations in DNA. The most common mutation is the

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 ...

cytosine Cytosine () ( symbol C or Cyt) is one of the four nucleobases found in DNA and RNA, along with adenine, guanine, and thymine (uracil in RNA). It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached (an ...

uracil Uracil () (symbol U or Ura) is one of the four nucleobases in the nucleic acid RNA. The others are adenine (A), cytosine (C), and guanine (G). In RNA, uracil binds to adenine via two hydrogen bonds. In DNA, the uracil nucleobase is replaced b ...

. UDG repairs these mutations. UDG is crucial in

DNA repair DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA da ...

, without it these mutations may lead 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 ...

. This entry represents various uracil-DNA glycosylases and related DNA glycosylases
EC
, such as uracil-DNA glycosylase,

thermophilic A thermophile is an organism—a type of extremophile—that thrives at relatively high temperatures, between . Many thermophiles are archaea, though they can be bacteria or fungi. Thermophilic eubacteria are suggested to have been among the earl ...

uracil-DNA glycosylase, G:T/U mismatch-specific DNA glycosylase (Mug), and single-strand selective monofunctional uracil-DNA glycosylase (SMUG1). Uracil DNA glycosylases remove

from DNA, which can arise either by spontaneous deamination of cytosine or by the misincorporation of dU opposite dA 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 living organisms acting as the most essential part for biological inheritanc ...

. The prototypical member of this family is E. coli UDG, which was among the first glycosylases discovered. Four different uracil-DNA glycosylase activities have been identified in mammalian cells, including UNG,

SMUG1 Single-strand selective monofunctional uracil DNA glycosylase is an enzyme that in humans is encoded by the ''SMUG1'' gene. SMUG1 is a glycosylase that removes uracil from single- and double-stranded DNA in nuclear chromatin, thus contributing to ...

, TDG, and

MBD4 Methyl-CpG-binding domain protein 4 is a protein that in humans is encoded by the ''MBD4'' gene. Structure Human MBD4 protein has 580 amino acids with a methyl-CpG-binding domain at amino acids 82–147 and a C-terminal DNA glycosylase domain at ...

. They vary in substrate specificity and subcellular localization. SMUG1 prefers single-stranded DNA as substrate, but also removes U from double-stranded DNA. In addition to unmodified uracil, SMUG1 can excise 5-hydroxyuracil, 5-hydroxymethyluracil and 5-formyluracil bearing an oxidized group at ring C5. TDG and MBD4 are strictly specific for double-stranded DNA. TDG can remove thymine glycol when present opposite guanine, as well as derivatives of U with modifications at carbon 5. Current evidence suggests that, in human cells, TDG and SMUG1 are the major enzymes responsible for the repair of the U:G mispairs caused by spontaneous cytosine deamination, whereas uracil arising in DNA through dU misincorporation is mainly dealt with by UNG. MBD4 is thought to correct T:G mismatches that arise from deamination of 5-methylcytosine to thymine in CpG sites. MBD4 mutant mice develop normally and do not show increased cancer susceptibility or reduced survival. But they acquire more C T mutations at CpG sequences in epithelial cells of the small intestine. The structure of human UNG in complex with DNA revealed that, like other glycosylases, it flips the target nucleotide out of the double helix and into the active site pocket. UDG undergoes a conformational change from an ‘‘open’’ unbound state to a ‘‘closed’’ DNA-bound state.Slupphaug G, Mol CD, Kavli B, Arvai AS, Krokan HE, Tainer JA. (1996). A nucleotide-flipping mechanism from the structure of human uracil–DNA glycosylase bound to DNA. 384: 87-92.

History

Lindahl was the first to observe repair of uracil in DNA. UDG was purified from ''Escherichia coli'', and this hydrolysed the N-

connecting the base to the deoxyribose sugar of the DNA backbone.

Function

The function of UDG is to remove mutations in DNA, more specifically removing uracil.

Structure

These

s have a 3-layer alpha/beta/alpha structure. The polypeptide topology of UDG is that of a classic alpha/beta protein. The structure consists primarily of a central, four-stranded, all parallel beta sheet surrounded on either side by a total of eight alpha helices and is termed a parallel doubly wound beta sheet.

Mechanism

Uracil-DNA glycosylases are DNA repair

s that excise

residue Residue may refer to: Chemistry and biology * An amino acid, within a peptide chain * Crop residue, materials left after agricultural processes * Pesticide residue, refers to the pesticides that may remain on or in food after they are applied ...

s from DNA by cleaving the N-glycosydic bond, initiating the

pathway. Uracil in DNA can arise either through the deamination of

to form mutagenic U:G mispairs, or through the incorporation of dUMP by DNA polymerase to form U:A

pairs Concentration, also known as Memory, Shinkei-suijaku (Japanese meaning "nervous breakdown"), Matching Pairs, Match Match, Match Up, Pelmanism, Pexeso or simply Pairs, is a card game in which all of the cards are laid face down on a surface and tw ...

. These aberrant uracil residues are genotoxic.

Localisation

eukaryotic Eukaryotes () are organisms whose Cell (biology), cells have a cell nucleus, nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the ...

cells, UNG activity is found in both the

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 Nucl ...

and the mitochondria.

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, cultu ...

UNG1 protein is transported to both the mitochondria and the

Conservation

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 calle ...

of uracil-DNA glycosylase is extremely well conserved in

bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of prokaryotic microorganisms. Typically a few micrometr ...

and eukaryotes as well as in herpes viruses. More distantly related uracil-DNA glycosylases are also found in

poxviruses ''Poxviridae'' is a family of double-stranded DNA viruses. Vertebrates and arthropods serve as natural hosts. There are currently 83 species in this family, divided among 22 genera, which are divided into two subfamilies. Diseases associated wit ...

. The N-terminal 77

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 a ...

s of UNG1 seem to be required for mitochondrial localization, but the presence of a mitochondrial transit

peptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. ...

has not been directly demonstrated. The most N-terminal conserved region contains an aspartic acid

which has been proposed, based on

X-ray An X-ray, or, much less commonly, X-radiation, is a penetrating form of high-energy electromagnetic radiation. Most X-rays have a wavelength ranging from 10 picometers to 10 nanometers, corresponding to frequencies in the range 30&nb ...

structures to act as a general base in the

catalytic mechanism Enzyme catalysis is the increase in the rate of a process by a biological molecule, an "enzyme". Most enzymes are proteins, and most such processes are chemical reactions. Within the enzyme, generally catalysis occurs at a localized site, calle ...

Family

There are two UDG families, named Family 1 and Family 2. Family 1 is active against uracil in ssDNA and dsDNA. Family 2 excise uracil from mismatches with

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 c ...

Glycosylases of oxidized bases

A variety of glycosylases have evolved to recognize oxidized bases, which are commonly formed by reactive oxygen species generated during cellular metabolism. The most abundant lesions formed at guanine residues are 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and

8-oxoguanine 8-Oxoguanine (8-hydroxyguanine, 8-oxo-Gua, or OH8Gua) is one of the most common DNA lesions resulting from reactive oxygen species modifying guanine, and can result in a mismatched pairing with adenine resulting in G to T and C to A substitutions ...

. Due to mispairing with adenine during replication, 8-oxoG is highly mutagenic, resulting in G to T transversions. Repair of this lesion is initiated by the bifunctional DNA glycosylase OGG1, which recognizes 8-oxoG paired with C. hOGG1 is a bifunctional glycosylase that belongs to the helix-hairpin-helix (HhH) family. MYH recognizes adenine mispaired with 8-oxoG but excises the A, leaving the 8-oxoG intact. OGG1 knockout mice do not show an increased tumor incidence, but accumulate 8-oxoG in the liver as they age. A similar phenotype is observed with the inactivation of MYH, but simultaneous inactivation of both MYH and OGG1 causes 8-oxoG accumulation in multiple tissues including lung and small intestine. In humans, mutations in MYH are associated with increased risk of developing colon polyps and colon cancer. In addition to OGG1 and MYH, human cells contain three additional DNA glycosylases, NEIL1, NEIL2, and NEIL3. These are homologous to bacterial Nei, and their presence likely explains the mild phenotypes of the OGG1 and MYH knockout mice.

Glycosylases of alkylated bases

This group includes E. coli AlkA and related proteins in higher eukaryotes. These glycosylases are monofunctional and recognize methylated bases, such as 3-methyladenine.

AlkA

AlkA refers to 3-methyladenine DNA glycosylase II.

Pathology

*DNA glycosylases involved in

(BER) 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 ...

and BRCA2 mutation carriers.

Epigenetic deficiencies in cancers

Epigenetic alterations (epimutations) in DNA glycosylase genes have only recently begun to be evaluated in a few cancers, compared to the numerous previous studies of epimutations in genes acting in other DNA repair pathways (such as MLH1 in mismatch repair and MGMT in direct reversal). Two examples of epimutations in DNA glycosylase genes that occur in cancers are summarized below.

MBD4 Methyl-CpG-binding domain protein 4 is a protein that in humans is encoded by the ''MBD4'' gene. Structure Human MBD4 protein has 580 amino acids with a methyl-CpG-binding domain at amino acids 82–147 and a C-terminal DNA glycosylase domain at ...

(methyl-CpG-binding domain protein 4) is a glycosylase employed in an initial step of base excision repair. MBD4 protein binds preferentially to fully methylated CpG sites. These altered bases arise from the frequent hydrolysis of cytosine to uracil (see image) and hydrolysis of 5-methylcytosine to thymine, producing G:U and G:T base pairs. If the improper uracils or thymines in these base pairs are not removed before DNA replication, they will cause transition

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 replication, DNA or viral repl ...

s. MBD4 specifically catalyzes the removal of T and U paired with guanine (G) within CpG sites. This is an important repair function since about 1/3 of all intragenic single base pair mutations in human cancers occur in CpG dinucleotides and are the result of G:C to A:T transitions. These transitions comprise the most frequent mutations in human cancer. For example, nearly 50% of somatic mutations of the tumor suppressor gene

p53 p53, also known as Tumor protein P53, cellular tumor antigen p53 (UniProt name), or transformation-related protein 53 (TRP53) is a regulatory protein that is often mutated in human cancers. The p53 proteins (originally thought to be, and often s ...

in colorectal cancer are G:C to A:T transitions within CpG sites. Thus, a decrease in expression of MBD4 could cause an increase in

carcinogenic A carcinogen is any substance, radionuclide, or radiation that promotes carcinogenesis (the formation of cancer). This may be due to the ability to damage the genome or to the disruption of cellular metabolic processes. Several radioactive subs ...

mutations. MBD4 expression is reduced in almost all colorectal

neoplasms A neoplasm () is a type of abnormal and excessive growth of tissue. The process that occurs to form or produce a neoplasm is called neoplasia. The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists ...

due to methylation of the promoter region of MBD4. Also MBD4 is deficient due to mutation in about 4% of colorectal cancers, A majority of histologically normal fields surrounding neoplastic growths (adenomas and colon cancers) in the colon also show reduced MBD4 mRNA expression (a field defect) compared to histologically normal tissue from individuals who never had a colonic neoplasm. This finding suggests that epigenetic silencing of MBD4 is an early step in colorectal

carcinogenesis Carcinogenesis, also called oncogenesis or tumorigenesis, is the formation of a cancer, whereby normal cells are transformed into cancer cells. The process is characterized by changes at the cellular, genetic, and epigenetic levels and abno ...

. In a Chinese population that was evaluated, the MBD4 Glu346Lys polymorphism was associated with about a 50% reduced risk of cervical cancer, suggesting that alterations in MBD4 is important in this cancer.

NEIL1

Nei-like (NEIL) 1 is a DNA glycosylase of the Nei family (which also contains NEIL2 and NEIL3). NEIL1 is a component of the DNA replication complex needed for surveillance of oxidized bases before replication, and appears to act as a “cowcatcher” to slow replication until NEIL1 can act as a glycosylase and remove the oxidatively damaged base. NEIL1 protein recognizes (targets) and removes certain oxidatively-damaged bases and then incises the

abasic site In biochemistry and molecular genetics, an AP site (apurinic/apyrimidinic site), also known as an abasic site, is a location in DNA (also in RNA but much less likely) that has neither a purine nor a pyrimidine base, either spontaneously or ...

via β,δ elimination, leaving 3′ and 5′ phosphate ends. NEIL1 recognizes oxidized pyrimidines, formamidopyrimidines,

thymine Thymine () ( symbol T or Thy) is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidi ...

residues oxidized at the methyl group, and both stereoisomers of thymine glycol. The best substrates for human NEIL1 appear to be the hydantoin lesions, guanidinohydantoin, and spiroiminodihydantoin that are further oxidation products of 8-oxoG. NEIL1 is also capable of removing lesions from single-stranded DNA as well as from bubble and forked DNA structures. A deficiency in NEIL1 causes increased mutagenesis at the site of an 8-oxo-Gua:C pair, with most mutations being G:C to T:A transversions. A study in 2004 found that 46% of primary gastric cancers had reduced expression of NEIL1

mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...

, though the mechanism of reduction was not known. This study also found that 4% of gastric cancers had mutations in the NEIL1 gene. The authors suggested that low NEIL1 activity arising from reduced expression and/or mutation of the NEIL1 gene was often involved in gastric carcinogenesis. A screen of 145 DNA repair genes for aberrant promoter methylation was performed on head and neck squamous cell carcinoma (HNSCC) tissues from 20 patients and from head and neck mucosa samples from 5 non-cancer patients. This screen showed that the NEIL1 gene had substantially increased hypermethylation, and of the 145 DNA repair genes evaluated, NEIL1 had the most significantly different frequency of methylation. Furthermore, the hypermethylation corresponded to a decrease in NEIL1 mRNA expression. Further work with 135 tumor and 38 normal tissues also showed that 71% of HNSCC tissue samples had elevated NEIL1 promoter methylation. When 8 DNA repair genes were evaluated in

non-small cell lung cancer Non-small-cell lung cancer (NSCLC) is any type of epithelial lung cancer other than small-cell lung carcinoma (SCLC). NSCLC accounts for about 85% of all lung cancers. As a class, NSCLCs are relatively insensitive to chemotherapy, compared to s ...

(NSCLC) tumors, 42% were hypermethylated in the NEIL1 promoter region. This was the most frequent DNA repair abnormality found among the 8 DNA repair genes tested. NEIL1 was also one of six DNA repair genes found to be hypermethylated in their promoter regions in colorectal cancer.

References

External links

* * {{Portal bar, Biology, border=no Protein domains DNA repair EC 3.2.2