Thermostable uracil-DNA glycosylase from Thermotoga maritima a member of a novel class of DNA repair enzymes

Sandigursky, Margarita; Franklin, William

doi:10.1016/s0960-9822(99)80237-1

Cited by 69 publications

(66 citation statements)

References 24 publications

(13 reference statements)

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“…The Pa-UDG sequence contains six cysteine residues of which four are totally conserved in the characterized T. maritima and Archeoglobus fulgidus Family 4 UDGs (7,8) and in many homologous archaeal and eubacterial (putative) UDG sequences (Fig. 5).…”

Section: Discussionmentioning

confidence: 99%

An Iron-Sulfur Cluster in the Family 4 Uracil-DNA Glycosylases

Hinks¹,

Evans²,

Miguel³

et al. 2002

Journal of Biological Chemistry

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The 25-kDa Family 4 uracil-DNA glycosylase (UDG) from Pyrobaculum aerophilum has been expressed and purified in large quantities for structural analysis. In the process we observed it to be colored and subsequently found that it contained iron. Here we demonstrate that P. aerophilum UDG has an iron-sulfur center with the EPR characteristics typical of a 4Fe4S high potential iron protein. Interestingly, it does not share any sequence similarity with the classic iron-sulfur proteins, although four cysteines (which are strongly conserved in the thermophilic members of Family 4 UDGs) may represent the metal coordinating residues. The conservation of these residues in other members of the family suggest that 4Fe4S clusters are a common feature. Although 4Fe4S clusters have been observed previously in Nth/MutY DNA repair enzymes, this is the first observation of such a feature in the UDG structural superfamily. Similar to the Nth/MutY enzymes, the Family 4 UDG centers probably play a structural rather than a catalytic role.Uracil-DNA glycosylases are ubiquitous DNA repair enzymes responsible for the excision of uracil bases from DNA as the first step in a base excision repair pathway. Uracil arises in DNA either as a result of the hydrolytic deamination of cytosine residues in G:C base pairs (1) or from incorporation of deoxyuridine monophosphate (instead of thymidine monophosphate) opposite adenine during DNA replication (2). If left uncorrected the former process would cause G:C to A:T transition mutations (1), whereas the latter may result in the disruption of specific regulatory DNA-protein interactions (3).Hyperthermophilic organisms are at especially high risk of DNA damage by cytosine deamination, which is significantly enhanced by elevated temperature (4). Because hyperthermophiles do not exhibit any greater susceptibility to this type of damage they presumably possess more effective repair enzymes (5). However, despite the detection of UDG 1 activity in several hyperthermophiles (6) no sequences homologous to the archetypal Escherichia coli ung-encoded enzyme were initially apparent in archaeal genomes. Subsequently, UDGs were identified in hyperthermophilic Eubacteria and Archaea (7-9) with more obvious homology to a second family of uracil base excision repair enzymes typified by the human thymine DNA glycosylase (TDG) (10) and the bacterial MUG (11). These G:T/U mismatch-specific enzymes (Family 2) are structurally and mechanistically related to the UNG-type UDGs (Family 1) (12, 13), and they unite the UNG-type and thermophile enzymes (Family 4) into a uracil-DNA glycosylase superfamily (14). Pyrobaculum aerophilum is a hyperthermophilic archaeon isolated from a boiling marine water hole and growing optimally at 100°C and pH 7.0 (15). A fosmid-based genomic map of the 1.7-Mb P. aerophilum genome was constructed and used to identify 474 putative genes (16), but no homologues of the UNG or MUG/TDG UDG families were initially identified. Following the identification of Tm-UDG (a novel UDG weakly related to E....

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Section: Discussionmentioning

confidence: 99%

An Iron-Sulfur Cluster in the Family 4 Uracil-DNA Glycosylases

Hinks¹,

Evans²,

Miguel³

et al. 2002

Journal of Biological Chemistry

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“…Pa-UDGa and hUNG2 belong to two distinct UDG families due to the low sequence similarity between them (31,38). In addition, Pa-UDGa is an olive green-colored protein (31) due to the presence of an iron-sulfur cluster (39), whereas hUNG2 is largely colorless.…”

Section: Expression and Partial Purification Of Two Putative Pcnamentioning

confidence: 99%

“…Although FEN homologs are present in Archaea and Eukarya (27), Pa-UDGa family members are found in Archaea and Eubacteria (38,40). The amino acid sequence alignments of the carboxyl-terminal regions of FEN and UDG homologs are shown in Fig.…”

Section: Expression and Partial Purification Of Two Putative Pcnamentioning

confidence: 99%

Direct Interaction between Uracil-DNA Glycosylase and a Proliferating Cell Nuclear Antigen Homolog in the CrenarchaeonPyrobaculum aerophilum

Yang

Chiang

Fitz‐Gibbon

et al. 2002

Journal of Biological Chemistry

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Proliferating cell nuclear antigen (PCNA) acts as a sliding clamp on duplex DNA. Its homologs, present in Eukarya and Archaea, are part of protein complexes that are indispensable for DNA replication and DNA repair. In Eukarya, PCNA is known to interact with more than a dozen different proteins, including a human major nuclear uracil-DNA glycosylase (hUNG2) involved in immediate postreplicative repair. In Archaea, only three classes of PCNA-binding proteins have been reported previously: replication factor C (the PCNA clamp loader), family B DNA polymerase, and flap endonuclease. In this study, we report a direct interaction between a uracil-DNA glycosylase (Pa-UDGa) and a PCNA homolog (Pa-PCNA1), both from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum (T opt ‫؍‬ 100°C). We demonstrate that the Pa-UDGa-Pa-PCNA1 complex is thermostable, and two hydrophobic amino acid residues on Pa-UDGa (Phe 191 and Leu 192 ) are shown to be crucial for this interaction. It is interesting to note that although Pa-UDGa has homologs throughout the Archaea and bacteria, it does not share significant sequence similarity with hUNG2. Nevertheless, our results raise the possibility that Pa-UDGa may be a functional analog of hUNG2 for PCNA-dependent postreplicative removal of misincorporated uracil.

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“…As their name implies, family 3 enzymes are preferentially active on uracil-containing ssDNA substrates. Family 4 enzymes have been identified in hyperthermophiles such as Thermotoga maritima (28), Archaeoglobus fulgidus (29), and Pyrobaculum aerophilum (6). These enzymes act on U opposite A or G and exhibit maximal activity at high temperatures.…”

mentioning

confidence: 99%

The Uracil DNA Glycosylase UdgB of Mycobacterium smegmatis Protects the Organism from the Mutagenic Effects of Cytosine and Adenine Deamination

et al. 2009

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Spontaneous hydrolytic deamination of DNA bases represents a considerable mutagenic threat to all organisms, particularly those living in extreme habitats. Cytosine is readily deaminated to uracil, which base pairs with adenine during replication, and most organisms encode at least one uracil DNA glycosylase (UDG) that removes this aberrant base from DNA with high efficiency. Adenine deaminates to hypoxanthine approximately 10-fold less efficiently, and its removal from DNA in vivo has to date been reported to be mediated solely by alkyladenine DNA glycosylase. We previously showed that UdgB from Pyrobaculum aerophilum, a hyperthermophilic crenarchaeon, can excise hypoxanthine from oligonucleotide substrates, but as this organism is not amenable to genetic manipulation, we were unable to ascertain that the enzyme also has this role in vivo. In the present study, we show that UdgB from Mycobacterium smegmatis protects this organism against mutagenesis associated with deamination of both cytosine and adenine. Together with Ung-type uracil glycosylase, M. smegmatis UdgB also helps attenuate the cytotoxicity of the antimicrobial agent 5-fluorouracil.

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Thermostable uracil-DNA glycosylase from Thermotoga maritima a member of a novel class of DNA repair enzymes

Cited by 69 publications

References 24 publications

An Iron-Sulfur Cluster in the Family 4 Uracil-DNA Glycosylases

An Iron-Sulfur Cluster in the Family 4 Uracil-DNA Glycosylases

Direct Interaction between Uracil-DNA Glycosylase and a Proliferating Cell Nuclear Antigen Homolog in the CrenarchaeonPyrobaculum aerophilum

The Uracil DNA Glycosylase UdgB of Mycobacterium smegmatis Protects the Organism from the Mutagenic Effects of Cytosine and Adenine Deamination

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