Uracil glycosylase

Cytosine deamination (G ) Spontaneous/ chemicals Uracil glycosylase AP endonuclease DNA polymerase DNA ligase... 

A uracil glycosylase recognizes and removes the uracil base, leaving an apyrimidinic (AP) site in the DNA strand. 

Bacteria generally have just one type of uracil DNA glycosylase, whereas humans have at least four types, with different specificities—an indicator of the importance of uracil removal from DNA. The most abundant human uracil glycosylase, UNG, is associated with the human replisome, where it eliminates the occasional U residue inserted in place of a T during replication. The deamination of C residues is 100-fold faster in single-stranded DNA than in double-stranded DNA, and... 

A method by which the non-mutant strand is destroyed vivo has been created by Kunkel (11,16,17). The DNA of interest is cloned into a normal M13 vector and transfected into cells which are deficient in the enzymes dUTPase (dut") and uracil glycosylase (ung ). This combination results in the production of a template ssDNA which contains a small number of deoxyuridine residues in place of the normal DNA component thymidine. The mutant oligonucleotide is then annealed as in the previous methods, extended and ligated. Since the (-)strand synthesized vitro does not contain any deoxyuridine residues, transfection into a cell line which possesses the two enzymes dUTPase and uracil glycosylase results in the destruction of the non-mutant strand, thus yielding mostly mutant progeny. 

Figure 36-23. Base excision-repair of DNA. The enzyme uracil DNA glycosylase removes the uracil created by spontaneous deamination of cytosine in the DNA. An endonuclease cuts the backbone near the defect then, after an endonuclease removes a few bases, the defect is filled in by the action of a repair polymerase and the strand is rejoined by a ligase. (Courtesy of B Alberts.)...

The great potential of QM/MM calculations has attracted much attention in the past decade and the number of studies published in recent years is now so large that it is not possible to cover them all here. Among recent QM/MM applications at different levels are para-hydroxybenzoate hydrolase [56, 74], citrate synthase [4-6, 52], uracil-DNA glycosylase [88], neuraminidase [89, 90], aldose reductase [91], human thrombin [92], glutathione S-transferases [57], and HIV protease [93]. 

Mismatch Repair. Mispairs that break the normal base-pairing rules can arise spontaneously due to DNA biosynthetic errors, events associated with genetic recombination and the deamination of methylated cytosine (Modrich, 1987). With the latter, when cytosine deaminates to uracil, an endonuclease enzyme, /V-uracil-DNA glycosylase (Lindahl, 1979), excises the uracil residue before it can pair with adenine at the next replication. However, 5-methyl cytosine deaminates to form thymine and will not be excised by a glycosylase. As a result, thymine exits on one strand paired with guanine on the sister strand, that is, a mismatch. This will result in a spontaneous point mutation if left unrepaired. For this reason, methylated cytosines form spontaneous mutation hot-spots (Miller, 1985). The cell is able to repair mismatches by being able to distinguish between the DNA strand that exists before replication and a newly synthesized strand. 

L4. Longo, M. C., Beminger, M. S., and Hartley, J. L., Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93, 125-128 (1990). 

Tl. Thornton, C. G., Hartley, J. L., et al., Utilizing uracil DNA glycosylase to control carryover contamination in PCR Characterization of residual UDG activity following thermal cycling. Biotechniques 13(2), 180-184 (1992). 

Uracil DNA glycosylases, for example, found in most cells, specifically remove from DNA the uracil that results from spontaneous deamination of cytosine. Mutant cells that lack this enzyme have a high rate of G=C to A=T mutations. This glycosylase does not remove uracil residues from RNA or thymine residues from DNA. The capacity to distinguish thymine from uracil, the product of cytosine deamination—necessary for the selective repair of the latter—may be one reason why DNA evolved to contain thymine instead of uracil (p. 293). 

Removal of abnormal bases Abnormal bases, such as uracil, which can occur in DNA either by deamination of cytosine or improper incorporation of dUTP instead of dTTP during DNA synthesis, are recognized by specific glycosylases that hydrolytically cleave them from the deoxyribose-phosphate backbone of the strand. This leaves an apyrimidinic site (or apurinic, if a purine was removed), referred to as an AP-site. 

Why is uracil-DNA glycosylase important in DNA repair Is it important for DNA replication ... 

Dinner AR, GM Blackburn, M Karplus (2001) Uracil-DNA glycosylase acts by substrate autocatalysis. Nature 413 (6857) 752-755... 

An elegant way to curb carryover of amplified DNA is to replace thymidine in the PCR with deoxyuridine, which can be eliminated from subsequent PCR amplifications by treating the reaction mixture exclusive of template DNA with uracil-DNA glycosylase, which is inactivated by heating before the PCR is done.26 However, because the digestion with uracil-DNA glycosylase only reduces amplification of contaminating... 

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