Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Structural Changes and Reaction Intermediates in the Catalytic Cycles of DNA Repair Enzymes
One of the main ways to repair damage to individual DNA bases is the base-excision repair (BER) pathway. The key enzymes in BER are DNA glycosylases, which recognize a variety of modified or mismatched bases and catalyze cleavage of the N-glycosidic bond to release the inappropriate base from the deoxyribose ring. Many glycosylases also catalyze a β-elimination (or lyase) reaction to effect strand scission after the base removal. Subsequent action of apurinic-apyrimidinic (AP) endonucleases and 3?-phosphodiesterases remove the remaining sugar fragment to produce a single-nucleotide gap with the proper 3?-OH and 5?-phosphate termini, a substrate for DNA polymerases. After the DNA polymerase adds the correct nucleotide, DNA ligase completes the BER process.
Bacterial Fpg and eukaryotic OGG1 are two proteins that share no sequence homology nor are they structurally similar. In spite of this, they both are able to remove 8-oxoguanine (oxoG), an abundant pre-mutagenic oxidized nucleobase, from DNA. Recently we have investigated conformational dynamics in several DNA repair enzymes, including E. coli Fpg and human OGG1, and in their DNA substrates by stopped-flow detection of tryptophan (Trp) and 2-aminopurine (2-aPu) fluorescence (1 - 5). In all cases, multiple transient changes in the fluorescence intensities of the enzymes and their DNA substrates were observed, indicating sequential conformational transitions in both macromolecules during the catalytic cycle. In this study, we have performed pre-steady-state quench-flow measurements of DNA cleavage by Fpg for substrates containing 8-oxoguanine or an AP site. There was a fast burst phase of product accumulation followed by a linear part, characteristic of the overall reaction rate limited by a post-incision step. The reaction progress was followed by ESI/MS after a reduction of the Schiff base intermediate with NaBH4, capturing the formation of two covalent enzyme?DNA intermediates: a cross-link between Fpg and C1? of the damaged nucleoside before the β-elimination step and a final conjugate of 4-oxo-2-pentenal with Fpg after the δ-elimination step. A comparison of the kinetics of DNA cleavage and covalent intermediate formation with the Trp fluorescence traces indicated that the regeneration of the free enzyme from its conjugate with 4-oxo-2-pentenal most likely occurs after the dissociation of the enzyme?product complex and limits the reaction under multiple-turnover conditions. The analysis of the Trp and 2-aPu fluorescence traces obtained for wild-type Fpg and its mutant forms F110W and F110A suggests that the search for damaged bases in DNA proceeds through intercalation of Phe-110 residue into the DNA helix. This step could initiate the eversion of the damaged deoxynucleoside into the catalytic center of enzyme. The fluorescence kinetics for Fpg interaction with DNA substrates containing a FRET donor/emitter or emitter/quencher pair (Cy3/Cy5 or fluorescein/dabcyl) shows that the eversion of damaged deoxynucleoside is combined with the introduction of a kink into the DNA helix.
The interaction of OGG1, the human functional counterpart of Fpg, with a 8-oxoguanine-containing substrate carrying a pair of FRET labels, Cy3/Cy5, led to a scission of the damaged DNA strand followed by separation of the donor/emitter pair and a resulting decrease in the fluorescence. The rate of this reaction coincides with the rate of the conformational transition in the OGG1 molecule detected through Trp fluorescence. Therefore, in contrast to Fpg, the release of OGG1 from the enzyme-product complex does not limit the overall rate of the process. In human cells, the repair of AP sites, either formed spontaneously or products of the glycosylase reaction, is initiated by a special AP endonuclease, APE1. This enzyme recognizes the AP sites in double-stranded DNA and makes a single nick in the phosphodiester backbone 5' to the AP site. We found that the rate of the AP site scission obtained for interaction with FRET-labeled DNA substrates was the same as the rate of the conformational transition in APE1 corresponding to the product release step. The data obtained for the APE1-N211A mutant indicated that the Asn-211 residue is not essential for AP site recognition and binding but specifically required for the efficient catalysis.
This work was supported by grants from the RFBR (07-04-00191) and Siberian Division of the Russian Academy of Sciences (28, 48).
References and Footnotes
N. A. Kuznetsov
Inst. of Chemical Biology