Conserved residues from all three monomers contribute to each of the three active sites within the dUTPase. Although even dUTPases from evolutionarily distant species possess similar structural and functional traits, in a few cases, a monomer dUTPase mimics the trimer structure through an unusual folding pattern.
Catalysis proceeds by way of an SN2 mechanism; a water molecule initiates in-line nucleophilic attack. Complete nuclear targeting requires approximately of the UNG2 N-terminal amino acids, although the unique N-terminal 44 amino acids contain the most important residues in the nuclear localization signal NLS. The N-terminal 90 amino acids of UNG2 are readily lost due to proteolysis by cellular proteases during purification, but the compact and fully active catalytic domain is even resistant to Proteinase K treatment.
The N-terminal part of UNG2 therefore probably forms an independent structural domain, or may be unstructured. UNG2 is located in the nucleoplasm and replication foci. These interactions take place in replication foci Otterlei et al. The specific role of UNG2 in removal of misincorporated uracil was demonstrated by the inhibition of immediate post-replicative removal of incorporated uracil in isolated nuclei by neutralizing anti-UNG antibodies Otterlei et al.
Incorporated uracil was eventually removed, however, indicating the presence in nuclei of a less efficient back-up system for removal of uracil Nilsen et al. UNG2 is ideally suited for removal of misincorporated uracil close to the rapidly moving replication fork because it has a turnover number — per min which is orders of magnitude higher than that of other uracil-DNA glycosylases.
This is likely due to the presence of back-up activities, among which SMUG1 may have a dominant role Nilsen et al. While these results are seemingly at odds with previous results in bacteria and yeast reviewed in Krokan et al. It is possible that mutation rates in a homogenous fraction of rapidly growing cells would be different.
In fact, inhibition of UNG2 in human glioma cells by expression of the protein inhibitor Ugi resulted in a threefold increase in spontaneous mutation rates in a shuttle vector, which is comparable to the average mutation rates in microorganisms Radany et al. Several lines of evidence indicate that human UNG may also play a role in viral infection. It could, however, be hypothesized that the latter utilizes cellular UNG to avoid misincorporation of uracil during synthesis of the replicative DNA intermediate.
Alternatively, the association of viral proteins with UNG might be important for their nuclear translocation. Further studies of the association between UNG and viral proteins may provide important insight into the biology of HIV-1 and other viruses. Our understanding of the individual molecular steps involved in enzymatic removal of uracil by the Ung proteins has largely evolved from analysis of the human, E. Crystal structures of HSV-1 Savva et al.
The three enzymes are structurally essentially identical, and substrate binding occurs in a highly conserved pocket providing shape and electrostatic complementarity to uracil and which is too narrow to accommodate purines. UNG2 is nine amino acids longer at the unique N-terminal end reviewed in Krokan et al. Correct orientation of the latter amide group is fixed by a cluster of water molecules at the base of the uracil-binding pocket Pearl, The contribution of both N as well as Y to specificity was also verified experimentally, as replacement of Asn with Asp in human UNG shifted the specificity of the mutant towards cytosine, while replacement of Y by the smaller Ala, Cys or Ser shifted the specificity towards thymine Kavli et al.
An important conclusion from the crystal structures was that uracil within the helical context of DNA could not be accommodated within the buried uracil-binding pocket. A conserved leucine human L , positioned directly above the uracil-binding pocket, was suggested as a candidate to assist the local melting of the DNA helix Mol et al. Furthermore, compression of the backbone flanking uracil was for the first time implicated in catalysis, assisted by extensive conformational changes in the enzyme upon formation of the productive complex.
In the LA mutant structure the uracil had dissociated, and the enzyme rebound to the product, an extrahelically positioned AP-site. This implied that the extrahelical conformation could be achieved even in the absence of the insertion of a hydrophobic side chain push.
More recent data, however, indicate that the function of the inserting leucine side chain may be more complex than merely pushing the uracil out of the double helix. When analysing kinetic parameters of E. Using stopped-flow experiments of E. When considering the energy contribution of each discrete event above to the overall catalytic reaction, one should bear in mind that DNA is a very heterogeneous substrate.
This is also reflected by the different efficiency whereby uracil is excized from different sequence contexts Eftedal et al. Recently the sequence specificity was re-examined using both single-stranded and duplex DNA substrates Bellamy and Baldwin, , and the authors conclude that the observed variations were not due to stability of the uracil itself within the DNA structure. Rather, local structure perturbations could affect uracil recognition, e.
Uracil binding induces considerable conformational changes in UNG, bringing key residues in optimal distances to favour catalysis Slupphaug et al. This is accompanied by large conformational strain induced upon the deoxyuridine Parikh et al. The developing negative charge at O2 is enzymatically stabilized by a neutral histidine E. Moreover, recent quantum- and molecular-mechanical calculations indicate that negative phosphate charges in the substrate itself may repel the anionic leaving group, and thus make a major contribution to the catalytic rate Dinner et al.
The authors suggest that such substrate autocatalysis may emerge as a general feature of DNA glycosylases. The observation that UNG had an higher affinity for the product AP-site than the actual substrate itself Parikh et al. Such rebinding has subsequently been observed for several DNA glycosylases Vidal et al.
Perhaps the least understood stage in the processing of uracil-DNA is how the glycosylases recognize these subtle lesions within vast stretches of DNA. This is further complicated by the fact that eukaryotic DNA is organized in complex nucleoprotein structures. In vitro , the UNG-proteins appear to function in both a processive and distributive fashion, depending on the salt concentration Bennett et al. When a uracil residue is encountered, the mechanism of initial recognition is not obvious.
Thus, the enzyme might instead flip every DNA base to probe against the specificity pocket. How the energetic cost of such a scanning mechanism is covered merits further investigation, however. SMUG1 removes uracil, as well as 5-hydroxymethyluracil 5-hmeU , from single- and double stranded DNA and is proposed to have an important role in removal of uracil resulting from cytosine deamination Nilsen et al. SMUG1 is not thought to have a role in removal of incorporated uracil and it does not accumulate in replication foci.
This procedure comprised in vitro expression from a library of cDNA, and electrophoretic mobility shift upon binding of damage-recognising protein to DNA containing modified nucleotides designed to target the active site of the glycosylases. The human counterpart was identified from EST databases Haushalter et al. Thus, genes for three out of four uracil-removing activities are located on chromosome The phylogeny of the uracil-DNA glycosylase genes will be discussed in more detail below.
Thus, the term single-strand selective is not entirely appropriate for the human enzyme. Furthermore, the xSMUG1 activity was not inhibited by the peptide inhibitor Ugi that efficiently inhibits both prokaryotic and eukaryotic uracil-DNA glycosylases belonging to the Ung -family. However, it may also be formed in a two-step reaction; first the methyl-group of 5-meC in CpG-contexts is oxidised to 5-hmeC, and subsequently this residue is deaminated to yield 5-hmeU Cannon Carlson et al.
Even in extracts from wild-type mice, mSMUG1 contributed a substantial fraction of the total UDG-activity under these assay conditions. In the presence of 7. The situation may be different in mouse. In conclusion, hSMUG1 is a non-abundant enzyme present in the nucleoplasm.
The major function may be in removal of 5hmU and deaminated cytosines, although it may be less important than UNG2 in the latter process, at least in human cells. This enzyme has a strict requirement for double-stranded substrates Baker et al.
Thus, there are apparently at least three different human enzymatic activities for removal of 5-hmeU from DNA. However, the major physiological role of TDG remains elusive. Interestingly, TDG may also function as a transcription factor Hardeland et al. It does not cleave the DNA backbone, and thus contains no lyase activity Neddermann et al. This is unexpected since TDG is generally very double-strand-specific, and since the substrate recognition, as deduced from data on the homologous bacterial protein MUG, involves interactions with both strands Barrett et al.
In the cases described above, the substrate is a base that has been modified. The base pairing in the DNA helix helps to determine its structure. Due to the different interactions between the bases, the dsDNA helix completes a full turn on its axis every ten bases.
Each base allows the helix to turn thirty-six degrees [2]. Adenine and guanine are both purine bases, this means that they have a double-ringed structure. Cytosine, uracil only present in RNA and thymine are pyrimidines and have single ringed structures. This page guides you through the biochemical steps of this process. Comments Close.
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