Uracil-DNA glycosylase

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Protein UNG PDB 1akz.png
Available structures
PDB Ortholog search: PDBe RCSB
Aliases UNG, DGU, HIGM4, HIGM5, UDG, UNG1, UNG15, UNG2, uracil DNA glycosylase
External IDs MGI: 109352 HomoloGene: 6585 GeneCards: 7374
RNA expression pattern
PBB GE UNG 202330 s at tn.png
More reference expression data
Species Human Mouse
RefSeq (mRNA)



RefSeq (protein)



Location (UCSC) Chr 12: 109.1 – 109.11 Mb Chr 5: 114.13 – 114.14 Mb
PubMed search [1] [2]
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Uracil-DNA glycosylase, also known as UNG or UDG, is a human gene[3] though orthologs exist ubiquitously among prokaryotes and eukaryotes and even in some DNA viruses. The first uracil DNA-glycosylase was isolated from Escherichia coli.[4]


The human gene encodes one of several uracil-DNA glycosylases. Alternative promoter usage and splicing of this gene leads to two different isoforms: the mitochondrial UNG1 and the nuclear UNG2.[3] One important function of uracil-DNA glycosylases is to prevent mutagenesis by eliminating uracil from DNA molecules by cleaving the N-glycosylic bond and initiating the base-excision repair (BER) pathway. Uracil bases occur from cytosine deamination or misincorporation of dUMP residues. After a mutation occurs, the mutagenic threat of uracil propagates through any subsequent DNA replication steps.[5] Once unzipped, mismatched guanine and uracil pairs are separated, and DNA polymerase inserts complementary bases to form a guanine-cytosine (GC) pair in one daughter strand and an adenine-uracil (AU) pair in the other.[6] Half of all progeny DNA derived from the mutated template inherit a shift from GC to AU at the mutation site.[6] UDG excises uracil in both AU and GU pairs to prevent propagation of the base mismatch to downstream transcription and translation processes.[6] With high efficiency and specificity, this glycosylase repairs more than 10,000 bases damaged daily in the human cell.[7] Human cells express five to six types of DNA glycosylases, all of which share a common mechanism of base eversion and excision as a means of DNA repair.[8]


UDG is made of a four-stranded parallel β-sheet surrounded by eight α-helices.[9] The active site comprises five highly conserved motifs that collectively catalyze glycosidic bond cleavage:[10][11]

  1. Water-activating loop: 63-QDPYH-67[11]
  2. Pro-rich loop: 165-PPPPS-169[9]
  3. Uracil-binding motif: 199-GVLLLN-204[9][10]
  4. Gly-Ser loop: 246-GS-247[9]
  5. Minor groove intercalation loop: 268-HPSPLS-273[9][10]


Glycosidic bond cleavage follows a “pinch-push-pull” mechanism using the five conserved motifs.[9]

Pinch: UDG scans DNA for uracil by nonspecifically binding to the strand and creating a kink in the backbone, thereby positioning the selected base for detection. The Pro-rich and Gly-Ser loops form polar contacts with the 3’ and 5’ phosphates flanking the examined base.[10] This compression of the DNA backbone, or “pinch,” allows for close contact between UDG and base of interest.[9]

Push: To fully assess the nucleotide identity, the intercalation loop penetrates, or pushes into, the DNA minor groove and induces a conformational change to flip the nucleotide out of the helix.[12] Backbone compression favors eversion of the now extrahelical nucleotide, which is positioned for recognition by the uracil-binding motif.[9] The coupling of intercalation and eversion helps compensate for the disruption of favorable base stacking interactions within the DNA helix. Leu272 fills the void left by the flipped nucleotide to create dispersion interactions with neighboring bases and restore stacking stability.[10]

Pull: Now accessible to the active site, the nucleotide interacts with the uracil binding motif. The active site shape complements the everted uracil structure, allowing for high substrate specificity. Purines are too large to fit in the active site, while unfavorable interactions with other pyrimidines discourage binding alternative substrates.[8] The side chain of Tyr147 interferes sterically with the thymine C5 methyl group, while a specific hydrogen bond between the uracil O2 carbonyl and Gln144 discriminates against a cytosine substrate, which lacks the necessary carbonyl.[8] Once uracil is recognized, cleavage of the glycosidic bond proceeds according to the mechanism below.

Step 1: Nucleophilic water attacks the C-N glycosidic bond (intercalation by Leu272 not shown for simplicity).
Step 2: Uracil intermediate leaves the DNA helix; hydrogen bonds in the active site stabilize the DNA backbone.
Step 3: Proton exchange generates free uracil.

The position of the residues that activate the water nucleophile and protonate the uracil leaving group are widely debated, though the most commonly followed mechanism employs the water activating loop detailed in the enzyme structure.[11][13] Regardless of position, the identities of the aspartic acid and histidine residues are consistent across catalytic studies.[9][10][11][13][14]

Laboratory Use[edit]

Uracil N-glycosylase (UNG) is an enzyme utilized in a powerful method to eliminate carryover PCR products in Real-Time PCR. This method modifies PCR products such that in a new reaction, any residual products from previous PCR amplifications will be digested and prevented from amplifying, but the true DNA templates will be unaffected.[15] While polymerase chain reaction (PCR) synthesizes abundant amplification products. Contamination of new PCRs with trace amounts of these products, called carry-over contamination, yields false positive results. Carry-over contamination from some previous PCR can be a significant problem, due both to the abundance of PCR products, and to the ideal structure of the contaminant material for re-amplification. However carry-over contamination can be controlled by the following two steps: (i) incorporating dUTP in all PCR products (by substituting dUPT for dTTP, or by incorporating uracil during synthesis of primers; and (ii) treating all subsequent fully preassembled starting reactions with uracil DNA glycosylase (UDG), followed by thermal inactivation of UDG. UDG cleaves the uracil base from the phosphodiester backbone of uracil-containing DNA, but has no effect on natural (i.e., thymine-containing) DNA. The resulting apyrimidinic sites block replication by DNA polymerases, and are very labile to acid/base hydrolysis. Because UDG does not react with dUTP, and is also inactivated by heat denaturation prior to the actual PCR, carry-over contamination of PCRs can be controlled effectively if the contaminants contain uracils in place of thymines.[5]


Uracil-DNA glycosylase has been shown to interact with RPA2.[16]


  1. ^ "Human PubMed Reference:". 
  2. ^ "Mouse PubMed Reference:". 
  3. ^ a b "Entrez Gene: UNG uracil-DNA glycosylase". 
  4. ^ Lindahl T, Ljungquist S, Siegert W, Nyberg B, Sperens B (May 1977). "DNA N-glycosidases: properties of uracil-DNA glycosidase from Escherichia coli". The Journal of Biological Chemistry. 252 (10): 3286–94. PMID 324994. 
  5. ^ a b Longo MC, Berninger MS, Hartley JL (Sep 1990). "Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions". Gene. 93 (1): 125–8. doi:10.1016/0378-1119(90)90145-H. PMID 2227421. 
  6. ^ a b c Pearl LH (Aug 2000). "Structure and function in the uracil-DNA glycosylase superfamily". Mutation Research. 460 (3-4): 165–81. doi:10.1016/S0921-8777(00)00025-2. PMID 10946227. 
  7. ^ Slupphaug G, Mol CD, Kavli B, Arvai AS, Krokan HE, Tainer JA (Nov 1996). "A nucleotide-flipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA". Nature. 384 (6604): 87–92. doi:10.1038/384087a0. PMID 8900285. 
  8. ^ a b c Lindahl T (Apr 2000). "Suppression of spontaneous mutagenesis in human cells by DNA base excision-repair". Mutation Research. 462 (2-3): 129–35. doi:10.1016/S1383-5742(00)00024-7. PMID 10767624. 
  9. ^ a b c d e f g h i Parikh SS, Putnam CD, Tainer JA (Aug 2000). "Lessons learned from structural results on uracil-DNA glycosylase". Mutation Research. 460 (3-4): 183–99. doi:10.1016/S0921-8777(00)00026-4. PMID 10946228. 
  10. ^ a b c d e f Zharkov DO, Mechetin GV, Nevinsky GA (Mar 2010). "Uracil-DNA glycosylase: Structural, thermodynamic and kinetic aspects of lesion search and recognition". Mutation Research. 685 (1-2): 11–20. doi:10.1016/j.mrfmmm.2009.10.017. PMC 3000906free to read. PMID 19909758. 
  11. ^ a b c d Acharya N, Kumar P, Varshney U (Jul 2003). "Complexes of the uracil-DNA glycosylase inhibitor protein, Ugi, with Mycobacterium smegmatis and Mycobacterium tuberculosis uracil-DNA glycosylases". Microbiology. 149 (Pt 7): 1647–58. doi:10.1099/mic.0.26228-0. PMID 12855717. 
  12. ^ Mol CD, Arvai AS, Slupphaug G, Kavli B, Alseth I, Krokan HE, Tainer JA (Mar 1995). "Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis". Cell. 80 (6): 869–78. doi:10.1016/0092-8674(95)90290-2. PMID 7697717. 
  13. ^ a b Schormann N, Grigorian A, Samal A, Krishnan R, DeLucas L, Chattopadhyay D (2007). "Crystal structure of vaccinia virus uracil-DNA glycosylase reveals dimeric assembly". BMC Structural Biology. 7: 45. doi:10.1186/1472-6807-7-45. PMC 1936997free to read. PMID 17605817. 
  14. ^ Savva R, McAuley-Hecht K, Brown T, Pearl L (Feb 1995). "The structural basis of specific base-excision repair by uracil-DNA glycosylase". Nature. 373 (6514): 487–93. doi:10.1038/373487a0. PMID 7845459. 
  15. ^ https://abcommunity.thermofisher.com/community/real-time_pcr/blog/2013/08/02/what-is-ung-and-how-does-it-work
  16. ^ Nagelhus TA, Haug T, Singh KK, Keshav KF, Skorpen F, Otterlei M, Bharati S, Lindmo T, Benichou S, Benarous R, Krokan HE (Mar 1997). "A sequence in the N-terminal region of human uracil-DNA glycosylase with homology to XPA interacts with the C-terminal part of the 34-kDa subunit of replication protein A". The Journal of Biological Chemistry. 272 (10): 6561–6. doi:10.1074/jbc.272.10.6561. PMID 9045683. 

Further reading[edit]