Orlando D. Schärer

Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily

BACKGROUND Reactive oxygen species, ionizing radiation, and other free radical generators initiate the conversion of guanine (G) residues in DNA to 8-oxoguanine (OG), which is highly mutagenic as it preferentially mispairs with adenine (A) during replication. Bacteria counter this threat with a multicomponent system that excises the lesion, corrects OG:A mispairs and cleanses the nucleotide precursor pool of dOGTP. Although biochemical evidence has suggested the existence of base-excision DNA repair proteins specific for OG in eukaryotes, little is known about these proteins. RESULTS Using substrate-mimetic affinity chromatography followed by a mechanism-based covalent trapping procedure, we have isolated a base-excision DNA repair protein from Saccharomyces cerevisiae that processes OG opposite cytosine (OG:C) but acts only weakly on OG:A. A search of the yeast genome database using peptide sequences from the protein identified a gene, OGG1, encoding a predicted 43 kDa (376 amino acid) protein, identical to one identified independently by complementation cloning. Ogg1 has OG:C-specific base-excision DNA repair activity and also intrinsic beta-lyase activity, which proceeds through a Schiff base intermediate. Targeted disruption of the OGG1 gene in yeast revealed a second OG glycosylase/lyase protein, tentatively named Ogg2, which differs from Ogg1 in that it preferentially acts on OG:G. CONCLUSIONS S. cerevisiae has two OG-specific glycosylase/lyases, which differ significantly in their preference for the base opposite the lesion. We suggest that one of these, Ogg1, is closely related in overall three-dimensional structure to Escherichia coli endonuclease III (endo III), a glycosylase/lyase that acts on fragmented and oxidatively damaged pyrimidines. We have recently shown that AlkA, a monofunctional DNA glycosylase that acts on alkylated bases, is structurally homologous to endo III. We have now identified a shared active site motif amongst these three proteins. Using this motif as a protein database searching tool, we find that it is present in a number of other base-excision DNA repair proteins that process diverse lesions. Thus, we propose the existence of a DNA glycosylase superfamily, members of which possess a common fold yet act upon remarkably diverse lesions, ranging from UV photoadducts to mismatches to alkylated or oxidized bases.

Crystal Structure of a Human Alkylbase-DNA Repair Enzyme Complexed to DNA: Mechanisms for Nucleotide Flipping and Base Excision

DNA N-glycosylases are base excision-repair proteins that locate and cleave damaged bases from DNA as the first step in restoring the genetic blueprint. The human enzyme 3-methyladenine DNA glycosylase removes a diverse group of damaged bases from DNA, including cytotoxic and mutagenic alkylation adducts of purines. We report the crystal structure of human 3-methyladenine DNA glycosylase complexed to a mechanism-based pyrrolidine inhibitor. The enzyme has intercalated into the minor groove of DNA, causing the abasic pyrrolidine nucleotide to flip into the enzyme active site, where a bound water is poised for nucleophilic attack. The structure shows an elegant means of exposing a nucleotide for base excision as well as a network of residues that could catalyze the in-line displacement of a damaged base from the phosphodeoxyribose backbone.

Recent progress in the biology, chemistry and structural biology of DNA glycosylases.

Since the discovery in 1974 of uracil DNA glycosylase (UDG), the first member of the family of enzymes involved in base excision repair (BER), considerable progress has been made in the understanding of DNA glycosylases, the polypeptides that remove damaged or mispaired DNA bases from DNA. We also know the enzymes that act downstream of the glycosylases, in the processing of abasic sites, in gap filling and in DNA ligation. This article covers the most recent developments in our understanding of BER, with particular emphasis on the mechanistic aspects of this process, which have been made possible by the elucidation of the crystal structures of several glycosylases in complex with their respective substrates, substrate analogues and products. The biological importance of individual BER pathways is also being appreciated through the inactivation of key BER genes in knockout mouse models. BioEssays 23:270–281, 2001.

Structural Basis for the Excision Repair of Alkylation-Damaged DNA

Base-excision DNA repair proteins that target alkylation damage act on a variety of seemingly dissimilar adducts, yet fail to recognize other closely related lesions. The 1.8 A crystal structure of the monofunctional DNA glycosylase AlkA (E. coli 3-methyladenine-DNA glycosylase II) reveals a large hydrophobic cleft unusually rich in aromatic residues. An Asp residue projecting into this cleft is essential for catalysis, and it governs binding specificity for mechanism-based inhibitors. We propose that AlkA recognizes electron-deficient methylated bases through pi-donor/acceptor interactions involving the electron-rich aromatic cleft. Remarkably, AlkA is similar in fold and active site location to the bifunctional glycosylase/lyase endonuclease III, suggesting the two may employ fundamentally related mechanisms for base excision.

The active site of the DNA repair endonuclease XPF–ERCC1 forms a highly conserved nuclease motif

XPF–ERCC1 is a structure‐specific endonuclease involved in nucleotide excision repair, interstrand crosslink repair and homologous recombination. So far, it has not been shown experimentally which subunit of the heterodimer harbors the nuclease activity and which amino acids contribute to catalysis. We used an affinity cleavage assay and located the active site to amino acids 670–740 of XPF. Point mutations generated in this region were analyzed for their role in nuclease activity, metal coordination and DNA binding. Several acidic and basic residues turned out to be required for nuclease activity, but not DNA binding. The separation of substrate binding and catalysis by XPF–ERCC1 will be invaluable in studying the role of this protein in various DNA repair processes. Alignment of the active site region of XPF with proteins belonging to the Mus81 family and a putative archaeal RNA helicase family reveals that seven of the residues of XPF involved in nuclease activity are absolutely conserved in the three protein families, indicating that they share a common nuclease motif.

Single-molecule manipulation of double-stranded DNA using optical tweezers: Interaction studies of DNA with RecA and YOYO-1

By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first bead is immobilized by the optical tweezers and the second by the micropipette. Movement of the micropipette allows manipulation and stretching of the DNA molecule, and the force exerted on it can be monitored simultaneously with the optical tweezers. We used this setup to study elongation of dsDNA by RecA protein and YOYO-1 dye molecules. We found that the stability of the different DNA-ligand complexes and their binding kinetics were quite different. The length of the DNA molecule was extended by 45% when RecA protein was added. Interestingly, the speed of elongation was dependent on the external force applied to the DNA molecule. In experiments in which YOYO-1 was added, a 10-20% extension of the DNA molecule length was observed. Moreover, these experiments showed that a change in the applied external force results in a time-dependent structural change of the DNA-YOYO-1 complex, with a time constant of approximately 35 s (1/e2). Because the setup provides an oriented DNA molecule, we determined the orientation of the transition dipole moment of YOYO-1 within DNA by using fluorescence polarization. The angle of the transition dipole moment with respect to the helical axis of the DNA molecule was 69 degrees +/- 3.

Crystal structure of a thwarted mismatch glycosylase DNA repair complex

The bacterial mismatch‐specific uracil‐DNA glycosylase (MUG) and eukaryotic thymine‐DNA glycosylase (TDG) enzymes form a homologous family of DNA glycosylases that initiate base‐excision repair of G:U/T mismatches. Despite low sequence homology, the MUG/TDG enzymes are structurally related to the uracil‐DNA glycosylase enzymes, but have a very different mechanism for substrate recognition. We have now determined the crystal structure of the Escherichia coli MUG enzyme complexed with an oligonucleotide containing a non‐hydrolysable deoxyuridine analogue mismatched with guanine, providing the first structure of an intact substrate‐nucleotide productively bound to a hydrolytic DNA glycosylase. The structure of this complex explains the preference for G:U over G:T mispairs, and reveals an essentially non‐specific pyrimidine‐binding pocket that allows MUG/TDG enzymes to excise the alkylated base, 3,N4‐ethenocytosine. Together with structures for the free enzyme and for an abasic‐DNA product complex, the MUG–substrate analogue complex reveals the conformational changes accompanying the catalytic cycle of substrate binding, base excision and product release.

DNA interstrand crosslinks: natural and drug-induced DNA adducts that induce unique cellular responses.

It is a testimony to the complexity of cancer that cytotoxic drugs are still a mainstay of therapeutic approaches to treat that disease. Perhaps even more paradoxical is the fact that cytotoxic therapy has its origin in the highly toxic mustard gas, which was developed for chemical warfare in the First World War. As with other crosslinking agents, such as mitomycin C, the chloroethylnitroso ureas and cisplatin, mustard gas and nitrogen mustards derived from it exert their cytotoxic action by forming DNA interstrand crosslinks (ICLs). ICLs covalently link two strands of DNA, thereby blocking vital aspects of DNA metabolism. Evidence that ICLs can also be formed endogenously, for example by the reaction of DNA with bifunctional lipid peroxidation products, has been obtained much more recently. ICLs are therefore something the cell has to deal with naturally, although these lesions are formed infrequently compared to adducts involving only one DNA strand. The importance of being able to process ICLs in healthy cells is underscored by the existence of the rare inherited human disorder Fanconi Anemia (FA), which is characterized by extreme sensitivity to ICL forming agents, but not other DNA damaging agents. Here I will review the formation, biological consequences and clinical importance of ICLs. Finally, I will discuss how progress in the chemical synthesis of ICLs will provide opportunities for studying the cellular responses to ICL forming agents.

Specific Binding of a Designed Pyrrolidine Abasic Site Analog to Multiple DNA Glycosylases

In the base excision DNA repair pathway, DNA glycosylases recognize damaged bases in DNA and catalyze their excision through hydrolysis of the N-glycosidic bond. Attempts to understand the structural basis for DNA damage recognition by DNA glycosylases have been hampered by the short-lived association of these enzymes with their DNA substrates. To overcome this problem, we have employed an approach involving the design and synthesis of inhibitors that form stable complexes with DNA glycosylases, which can then be studied biochemically and structurally. We have previously reported that double-stranded DNA containing a pyrrolidine abasic site analog (PYR) forms an extremely stable complex with the DNA glycosylase AlkA and potently inhibits the reaction catalyzed by the enzyme (Schärer, O. D., Ortholand, J.-Y., Ganesan, A., Ezaz-Nikpay, K., and Verdine, G. L. (1995) J. Am. Chem. Soc.117, 6623–6624). Here we investigate the interaction of this inhibitor with a variety of additional DNA glycosylases. With the exception of uracil DNA glycosylase all the glycosylases tested bind specifically to PYR-containing oligonucleotides. By comparing the interaction of DNA glycosylases with PYR and the structurally related tetrahydrofuran abasic site analog, we assess the importance of the positively charged ammonium group of the pyrrolidine in binding to the active site of these enzymes. Such a general inhibitor of DNA glycosyases provides a valuable tool to study stable complexes of these enzymes bound to substrate-like molecules.

The spacer region of XPG mediates recruitment to nucleotide excision repair complexes and determines substrate specificity

XPG has structural and catalytic roles in nucleotide excision repair (NER) and belongs to the FEN-1 family of structure-specific nucleases. XPG contains a stretch of over 600 amino acids termed the “spacer region” between the conserved N- and I-nuclease regions. Its role is unknown, and it is not similar to any known protein. To investigate its possible functions, we generated and analyzed several deletion mutants of XPG. The spacer region is not required for endonuclease activity, but amino acids 111–550 contribute to the substrate specificity of XPG, and they are required for interaction with TFIIH and for NER activity in vitro and in vivo. Deletion of residues 184–210 and 554–730 leads only to a partial defect in NER activity and a weakened interaction with TFIIH. XPGΔ184–210 and XPGΔ554–730 are not observed at sites of local UV damage in living cells by immunofluorescence, suggesting that the weakened interaction between XPG and TFIIH results in an NER reaction with altered kinetics. This study demonstrates that the N-terminal portion of the spacer region is particularly important for NER progression by mediating the XPG-TFIIH interaction and XPG substrate specificity.