DNA polymerase eta (Pol eta) is a eukaryotic lesion bypass polymerase that helps organisms to survive exposure to ultraviolet (UV) radiation, and tumor cells to gain resistance against cisplatin-based chemotherapy. It allows cells to replicate across cross-link lesions such as 1,2-d(GpG) cisplatin adducts (Pt-GG) and UV-induced cis-syn thymine dimers. We present structural and biochemical analysis of how Pol eta copies Pt-GG-containing DNA. The damaged DNA is bound in an open DNA binding rim. Nucleotidyl transfer requires the DNA to rotate into an active conformation, driven by hydrogen bonding of the templating base to the dNTP. For the 3'dG of the Pt-GG, this step is accomplished by a Watson-Crick base pair to dCTP and is biochemically efficient and accurate. In contrast, bypass of the 5'dG of the Pt-GG is less efficient and promiscuous for dCTP and dATP as a result of the presence of the rigid Pt cross-link. Our analysis reveals the set of structural features that enable Pol eta to replicate across strongly distorting DNA lesions.

The Geobacillus stearothermophilus splG gene encodes a thermophilic spore photoproduct lyase (SplG) that belongs to the family of radical S-adenosylmethionine (AdoMet) enzymes. The aerobically purified apo-SplG forms a homodimer, which contains one [4Fe-4S] cluster per monomer unit after reconstitution to the holoform. Formation of the [4Fe-4S] cluster was proven by quantification of the amount of iron and sulfur per homodimer and by UV and EPR spectroscopy. The UV spectrum features a characteristic absorbance at 420 nm typical for [4Fe-4S] clusters, and the EPR data were found to be identical to those of other proteins containing an [4Fe-4S] ؉ center. Probing of the activity of the holo-SplG with oligonucleotides containing one spore photoproduct lesion at a defined site proved that the enzyme is able to turn over substrate. In addition to repair, we observed cleavage of AdoMet to generate 5-deoxyadenosine. In the presence of aza-AdoMet the SplG is completely inhibited, which provides direct support for the repair mechanism.Spores of various Bacillus and Clostridium species are extremely resistant to harsh physical, chemical, and biological conditions allowing them to survive even under extreme conditions (1, 2). The oldest known viable spore was discovered from a Bacillus species, designated 2-9-3, in a 250 million-yearold salt crystal from the Permian Salado Formation (3). The resistance of spores from Geobacillus stearothermophilus toward heat is even so high that the survival of the organism during heat sterilization is used as a bioindicator for insufficient heat treatment (4).Particularly noteworthy is the unusually high stability of spores in the presence of UV light. For example under typical UV sterilization conditions, only about 70% of thermophilic G. stearothermophilus spores are inactivated. Under the same conditions, typical pathogens such as herpes simplex or polio viruses are fully destroyed (5, 6). In addition, UV irradiation of spores gives rise to different DNA lesions (7). Although in normal cells mostly cyclobutane pyrimidine dimers and (6-4) lesions (8) are formed, in spores the unusual photoproduct 5-thyminyl-5,6-dihydrothymine (SP), 2 depicted in Scheme 1 (7, 9), is exclusively generated (10). These differences in the photoreactivity may be because of an unusual packing of the DNA in spores (1, 11, 12) and the high amounts of dipicolinic acid (DPA) present in spores (13).During germination, the SP lesion is repaired either by the general nucleotide excision repair pathway (14, 15) or by a single enzyme, called spore photoproduct lyase, which is able to split the SP lesions directly back into two thymidines. Recent studies by Nicholsen et al. (16) and Broderick and co-workers (17) performed with the SP-lyase from Bacillus subtilis showed that the enzyme requires S-adenosylmethionine (AdoMet) as a cofactor for repair. A detailed sequence comparison, spectroscopic studies (18,19), and a recent labeling experiment (17) all provide evidence that the SplG is a member of the radical AdoMet enz...

The spore photoproduct lyase is a Fe-S/AdoMet DNA repair enzyme, which directly repairs spore lesions, induced by UV irradiation of spores, using an unknown radical mechanism. The air sensitive radical SAM enzyme was for the first time challenged with synthetically pure substrates. It was found that the enzyme recognizes a synthetic 5S-configured spore lesion without the central phosphodiester bond. The 5R-configured lesion is in contrast to current belief not a substrate.

Investigation of the DNA repair process performed by the spore photoproduct (SP) lyase repair enzyme is strongly hampered by the lack of defined substrates needed for detailed enzymatic studies. The problem is particularly severe because the repair enzyme belongs to the class of strongly oxygen-sensitive radical (S)-adenosylmethionine (SAM) enzymes, which are notoriously difficult to handle. We report the synthesis of the spore photoproduct analogues 1 a and 1 b, which have open backbones and are diastereoisomers. In order to solve the problem of stereochemical assignment, two further derivatives 2 a and 2 b with closed backbones were prepared. The key step of the synthesis of 2 a/b is a metathesis-based macrocyclization that strongly increases the conformational rigidity of the synthetic spore photoproduct derivatives. NOESY experiments of the cyclic isomers furnished a clear cross-peak pattern that allowed the unequivocal assignment of the stereochemistry. The results were transferred to the data for isomers 1 a and 1 b, which were subsequently used for enzymatic-repair studies. These studies were performed with the novel spore photoproduct lyase repair enzyme from Geobacillus stearothermophilus. The studies showed an accordance with a recent investigation performed by us with the spore photoproduct lyase from Bacillus subtilis, in that only the S isomer 1 a is recognized and repaired. The ability to prepare a defined functioning substrate now paves the way for detailed enzymatic studies of the SP-lyase lesion recognition and repair process.

The oxidative DNA lesion, FaPydG rapidly anomerizes to form a mixture of the alpha and beta anomer. To investigate the mutagenic potential of both forms, we prepared stabilized bioisosteric analogues of both configurational isomers and incorporated them into oligonucleotides. These were subsequently used for thermodynamic melting-point studies and for primer-extension experiments. While the beta compound, in agreement with earlier data, prefers cytidine as the pairing partner, the alpha compound is not able form a stable base pair with any natural base. In primer-extension studies with the high-fidelity polymerase Bst Pol I, the polymerase was able to read through the lesion. The beta compound showed no strong mutagenic potential. The alpha compound, in contrast, strongly destabilized DNA duplexes and also blocked all of the tested DNA polymerases, including two low-fidelity polymerases of the Y-family.

An acetophenone containing PNA-based reagent was designed for the direct and site-specific synthesis of a cis-syn thymidine dimer lesion in genomic DNA.

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