An Iron–Sulfur Cluster Loop Motif in the Archaeoglobus fulgidus Uracil–DNA Glycosylase Mediates Efficient Uracil Recognition and Removal

Abstract

The Family 4 Uracil DNA glycosylase from the hyperthermophilic organism Archaeoglobus fulgidus (AFUDG) is responsible for the removal of uracil in DNA as the first step in the Base Excision Repair (BER) pathway. AFUDG contains a large solvent-exposed peptide region containing an alpha helix and loop anchored on each end via ligation of two cysteine thiolates to a [4Fe-4S]2+ cluster. We propose that this region plays a similar role in DNA damage recognition as a smaller iron-sulfur cluster loop (FCL) motif in the structurally unrelated BER glycosylases MutY and Endonuclease III, and therefore refer to this region as the “pseudo-FCL” in AFUDG. In order to evaluate the importance of this region, three positively charged residues (Arg 86, Arg 91, Lys 100) and the anchoring Cys residues (Cys 85, Cys 101) within this motif were replaced with alanine and the effects of these replacements on uracil excision in single and double-stranded DNA were evaluated. These results show that this region participates and allows for efficient recognition and excision of uracil within DNA. Notably, R86A AFUDG exhibited reduced activity for uracil removal only within double-stranded DNA, suggesting an importance in duplex disruption and extrusion of the base as part of the excision process. In addition, mutation of the [4Fe-4S]2+ cluster cysteine ligands at the ends of the pseudo-FCL to alanine reduced the uracil excision efficiency, suggesting the importance of anchoring the loop via coordination to the cluster. In contrast, K100A AFUDG exhibited enhanced uracil excision activity, providing evidence for the importance of the loop conformation and flexibility. Taken together, the results herein provide evidence that the pseudo-FCL motif is involved in DNA binding and catalysis, particularly in duplex DNA contexts. This work underscores the requirement of an ensemble of interactions, both distant and in proximity to the damaged site, for accurate and efficient uracil excision.

The pH optimum of native uracil-DNA glycosylase of Archaeoglobus fulgidus compared to recombinant enzyme indicates adaption to cytosolic pH.

“…Afung is one of the most studied family 4 UDGs (Knaevelsrud et al, 2010;Knaevelsrud et al, 2001;Sandigursky & Franklin, 2000), and is, due to the presence of a [4Fe-4S] 2+ cofactor, redox-active when bound to DNA. Thus, together with some other DNA glycosylases, Afung has been studied in the context of an interesting theory on the involvement of redox activity and DNA-mediated electron transfer in DNA damage recognition (Boal et al, 2005;Engstrom et al, 2012). Our precise measurement of the optimal pH for Afung in cell extracts (nAfung) compared to purified enzyme (rAfung; Fig.…”

“…UDG activity in hyperthermophilic microor-ganisms was first reported in 1996 (Koulis et al, 1996). The hyperthermophilic archaeon Archaeoglobus fulgidus, a strict anaerobe growing optimally at 83°C (Stetter, 1992), contains a family 4 type of UDG named Afung, which has been cloned and over-expressed in E. coli followed by biochemical characterization (Engstrom et al, 2012;Knaevelsrud et al, 2001;Sandigursky & Franklin, 2000). Recently, immunodepletion of UDG activity present in archaeal cell extract showed that Afung is the principal and probably the only UDG in A. fulgidus (Knaevelsrud et al, 2010).…”

Redox Signaling through DNA

“…Many of these proteins, however, do not require the cluster for stability or protein folding ,. There is evidence that a stable and intact cluster is necessary for substrate recognition and turnover but not for a role in catalysis, suggesting a regulatory role for the cluster in enzymatic function ,,. One might furthermore expect, given the possibility of Fenton chemistry with the iron‐sulfur clusters near DNA, that a more redox‐inert structural motif would better stabilize DNA binding …”

Fe-S Proteins Acting as Redox Switch: New Key Actors of Cellular Adaptive Responses

“…The crystal structures of these enzymes bound to DNA substrate reveal that the cluster is necessary to properly position conserved basic residues from the FCL to interact with the DNA phosphate backbone [89][90][91]. The importance of these residues of FCL in enzyme binding to substrate and activity was confirmed by sitedirected mutagenesis [74,92,93]. Thus, the Fe-S cluster clearly plays, at least, a structural role in these Fe-S clustercontaining glycosylases.…”