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. O 6 meG in the template may direct incorporation of either C or T. The rate-limiting step in the mechanism in vitro is the release of the product e. Recent findings indicate, however, that the AP-site binding capacity of TDG might be subject to regulation. Hardeland et al.
The authors propose that TDG binds its substrate in the unmodified state, and that subsequent to catalysis, sumoylation allows detachment from the product AP-site. Interestingly, a sumoylation consensus site is also present in MBD4 MED1 , but the functional implications of this remain to be established.
However, they appear to have very similar structures. The structure of the bacterial homologue MUG has been solved Barrett et al. Both enzymes are traversed by a DNA-binding groove connecting to a uracil-binding pocket, which in the case of MUG is less tailor-made for uracil.
Two active site motifs are conserved. These are identical in human, E. In both cases, the base can only be accommodated in the catalytic pocket after the nucleotide is flipped out of the helix. In the corresponding position in MUG, the amino acid is Gly20 which does not represent a major barrier to thymine.
However, the Ser side chain is free to rotate. The smaller side chain of Ala represents an even smaller barrier to binding of T. These extensions may be involved in subcellular sorting and protein—protein interactions, although this has not yet been demonstrated. RAR and RXR are intracellular receptors that after binding of ligand function as transcription factors. Furthermore, TDG has been shown to be a strong repressor of thyroid transcription factor-1 TTF-1 transcriptional activity Missero et al.
Whether the transcription-associated activity of TDG is modulated by sumoylation is presently not known. This, however merits further investigation, since several transcription factors appear to be directly or indirectly regulated by sumoylation reviewed by Muller et al.
In addition, it may function as a transcription factor. Which of these functions is the most important remains unclear. MBD4 may also have an additional role in mismatch repair through its interaction with MLH1 reviewed in Bellacosa, Furthermore, there is significant evidence associating defects in the MBD4 gene with human cancer reviewed in Hardeland et al.
The gene for MBD4 has been mapped to position 3q It is thus the only one of the four uracil-removing proteins that is not located to human chromosome 12 and it is apparently unrelated to the other uracil-DNA glycosylases. MBD4 was also found to interact with the human mismatch repair protein MLH1 in a yeast two-hybrid system.
The amino acid protein contains an N-terminal methyl-binding domain, MBD residues 82— and a C-terminal glycosylase domain residues — with homology to E. A CpG sequence context was preferred, but not absolutely required since mismatches in other sequences were processed at lower rates Hendrich et al.
It was therefore proposed that the function of MBD4 is to counteract the mutagenic effects of deamination of 5-meC to thymine. In this function, the MBD domain may be responsible for the binding specificity and the glycosylase domain for catalysis Hendrich and Bird, It might therefore contribute to active changing of the methylation status in cells Zhu et al.
This, as well as the mismatch repair function, are potential functions that could be relevant to the possible role of MBD4 in cancer prevention reviewed by Bellacosa, Furthermore, MBD4 mutations were reported in 14 cases of human cancer with microsatellite instability, but not in tumour without microsatellite instability. All of the mutations were deletions of A one or two in a polyA track, resulting in frame shift and generation of a stop codon.
These findings suggest that MBD4 may be important in prevention of cancer. In addition, Pa -UDGb was able to remove hypoxanthine, and thus is the first member of the UDG superfamily able to remove both pyrimidines and purines Sartori et al. The authors thus proposed that Pa -UDGb represented a fifth UDG family, that evolved in organisms living at elevated temperatures to counteract the mutagenic threat of both cytosine and adenine deamination.
The classification of DNA glycosylases into superfamilies, can be based on characteristic sequence motifs as defined in the Pfam database Bateman et al. MBD4 belongs to the HhH—GPD family, members of which remove a large variety of lesions, including uracil, oxidised bases and certain mismatches, particularly A mismatched to G or 8-oxoG.
The phylogenetic distribution of DNA repair genes has been discussed in detail by Eisen and Hanawalt , while Aravind and Koonin have specifically analysed the UDG superfamily.
The HhH—GPD glycosylases are widespread in both Archaea, bacteria and eukaryotes, and are believed to represent a very ancient gene family. However, the UDG superfamily shows a more non-even distribution pattern. It is distantly related to the AUDG gene family, which is mainly found in Archaea and bacteria, and it seems likely that they share an ancient ancestor.
SMUG1 has so far been found only in some eukaryotes. Based on the conservation pattern in the minor-groove intercalation loop it was suggested that SMUG1 may have evolved from an UNG-like enzyme by rapid divergence, possibly to meet special requirements for repair in multicellular animals Aravind and Koonin, These studies are based on relatively few sequences with low similarity.
This makes alignment and analysis difficult, and the interpretation of these data should be done with caution. The origin of SMUG1 therefore remains an open question. UNG is very widespread in bacteria, and has also been identified in most eukaryotes. It has been suggested that UNG was introduced into eukaryotes by horizontal gene transfer Aravind and Koonin, , possibly from the mitochondrial genome Eisen and Hanawalt, UNG is also the only of these gene families that is found in a large number of viruses, indicating another possible mechanism for horizontal gene transfer.
A Blast search Altschul et al. Sequence comparison of the different gene families within the UDG superfamily identifies several conserved sequence motifs, indicating a common 3D-fold for all UDG-type proteins. It therefore seems realistic to assume that all UDG-type proteins share this common fold. The essential part of the proline-rich motif that is in direct contact with the DNA backbone is structurally conserved in MUG.
However, the actual region seems to be well conserved in most sequences, the variation is mainly with respect to the specific residues found at each position. The motif contributes to uracil recognition by hydrogen bonding to polar atoms of the uracil ring. In the uracil binding pocket there is also a favourable stacking interaction between uracil and a well-conserved phenylalanine residue found between the water-activating and the proline-rich motifs, in addition to the tyrosine from the water-activating motif mentioned above.
This motif is also involved in DNA interaction. In particular the glycine is well conserved, probably because a side chain at this position would interfere with the close contact between the protein and the DNA backbone.
This motif shows some variation, but the histidine and the first proline are conserved in most sequences, except in SMUG1. The histidine is one of the active site residues, and forms hydrogen bond with uracil. This architecture family is a very large one, with 70 topologies listed in CATH. The 3D structure for MBD4 is not known. However, structures are available for several other members of this superfamily, and it can be assumed that important structural features are conserved.
The HhH—GPD-fold consists of a four-helix bundle domain and a six-helix barrel domain, with the active site and the HhH motif located at the interface between these domains. Whereas most DNA-binding proteins seem to use a charged surface rich in lysine and arginine residues to bind backbone phosphates, the DNA binding surface of OGG1 is nearly charge neutral.
UNG-proteins are highly selective for uracil, but remove 5-fluorouracil and certain oxidised pyrimidines with very low efficiency Krokan et al.
The efficient removal of uracil from single-stranded DNA is puzzling since it leaves a non-informative lesion without the information in a complementary strand. Single-stranded DNA is probably mainly found temporarily in transcribed genes and very close to the replication fork. Abasic sites resulting from uracil-removal in single-stranded DNA at the replication fork could be handled by at least three different mechanisms; i regression of the replication fork and repair by short patch or long patch BER, ii recombination repair using the old strand at the other side of the fork, iii translesion DNA synthesis.
Regression of a replication fork stalled at a single-strand lesion is well established in E. It may in principle apply to all types of lesions that stall the replication fork, including abasic sites Robu et al. Recombination using information from the sister chromatid at stalled replication forks Gruss and Michel, , as well as translesion synthesis across abasic sites are well established processes in bacteria. Interestingly, repair of abasic sites in chromosomal DNA in E.
Therefore, abasic sites resulting from the action of UNG at the replication fork is possibly unlikely to be dealt with by BER alone. For more comprehensive overviews, the reader should consult other recent reviews Dogliotti et al. The presumed major track is the short patch pathway.
The alternative long patch pathway largely uses replication proteins Dogliotti et al. As shown in Figure 2 , uracil in DNA may be present in different positions relative to a replication fork, and in addition the sequence context may vary. But the pathogens that cause disease are increasingly developing resistance to the ZCCHC4 influences cell Although, other nucleic acid-like polymers are known, yet much remains unknown regarding possible RNA, or ribonucleic acid, is a molecule that plays a central role in the function of The modification is apparently attached to molecules only when cells are under stress, and is rapidly removed Using a novel technique, researchers have been able What Makes Us Human?
Stem cell researchers have now found a previously overlooked The researchers hypothesize that a lower channel density may have The results illuminate one of the Researchers have shown that it is possible to identify individual proteins with single-amino acid Print Email Share.
Most Popular Stories. Just a Game? You Need a Chickadee Brain.
0コメント