What does Glycosylase do in DNA?
DNA glycosylases play a key role in the elimination of such DNA lesions; they recognize and excise damaged bases, thereby initiating a repair process that restores the regular DNA structure with high accuracy.
What does uracil glycosylase do?
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.
How does base excision repair work?
Base excision repair (BER) corrects small base lesions that do not significantly distort the DNA helix structure. It is initiated by a DNA glycosylase that recognizes and removes the damaged base, leaving an abasic site which is further processed by short-patch repair or long-patch repair.
What is adenine Glycosylase?
Adenine DNA glycosylase catalyzes the glycolytic removal of adenine from the promutagenic A·oxoG base pair in DNA.
Where is Glycosylase found?
The sequence of uracil-DNA glycosylase is extremely well conserved in bacteria and eukaryotes as well as in herpes viruses. More distantly related uracil-DNA glycosylases are also found in poxviruses.
What is the purpose of UNG treatment?
The biological function is to remove uracil—normally found in RNA—from DNA, creating free uracil and alkali-sensitive apyrimidic sites in DNA [2,6]. UNG removes uracil incorporated into single- and double-stranded DNA by catalyzing hydrolysis in the N-glycosylic bond between uracil and sugar [7], 2008).
How does the DNA glycosylase identify a base lesion?
DNA glycosylases recognize and remove damaged bases from DNA by cleaving the base–sugar (N-glycosylic) bond, and downstream base excision repair enzymes restore the correct nucleotide.
How does a Glycosylase work?
Is 5 Bromouracil a base analog?
5-Bromouracil (BrU) is a base analogue of thymine (T) which can be incorporated into DNA. It is a well-known mutagen, causing transition mutations by mispairing with guanine (G) rather than pairing with adenine (A) during replication.
How does DNA glycosylase initiate Ber?
DNA glycosylases initiate BER by cleaving the glycosidic bond between damaged nitrogenous bases and the pentose moiety [174–176]. Endonuclease III, which is the glycosylase that functions in removing oxidized pyrimidines from duplex DNA, was found to contain a [4Fe-4S] cluster.
What is the function of the C-terminal glycosylase domain?
The C-terminal glycosylase domain excises uracil, thymine (also known as 5-methyluracil) and 5-hydroxymethyluracil (5hmU) paired with guanine, which forms deamination linked mismatches. MBD4 catalytic inactive mutant (Asp534Asn for mouse, Asp560Ala for human) and substrate DNA crystal structures clarifies how MBD4 recognizes substrates22,23.
What is the reaction volume of a generalised glycosylase assay?
Glycosylase assay reactions are carried out in 100 μL volumes total following enzyme addition to the reaction mix ( Table 4 ). The positive and negative controls (excess enzyme and no enzyme, respectively) can be carried out in as little as 20 μL reaction volumes.
Is the glycosylase domain of MBD4 bound to thymine?
Biochemical and structural characterization of the glycosylase domain of MBD4 bound to thymine and 5-hydroxymethyuracil-containing DNA. Nucleic Acids Res. 2012;40:9917–9926.