Book of Abstracts: Albany 2007
June 19-23 2007
Human AP endonuclease 1: the Dynamics of Conformational Changes
In the process of cell metabolism, genomic DNA is harmed by free radicals, mutagens, and ionizing radiation. The damage in DNA results in carcinogenesis, aging, and cell death. Apurinic/apyrimidinic (AP) sites, which arise by hydrolysis of N-glycosidic bonds, belong to the most common DNA lesions. Repair of such lesions is accomplished in different ways including base excision repair (BER). The crucial enzyme of this repair pathway in human cells is AP endonuclease 1 (Ape1) which recognizes AP sites in dsDNA and makes a single nick in the phosphodiester backbone 5' to the AP site. Detailed investigation of the kinetics of repair processes is very important for understanding the fundamental bases of this process.
Many enzymatic reactions are accompanied by conformational changes both in the enzyme and the substrate molecules (1-3). Conformational transitions in a protein molecule can be followed by changes in the intensity of fluorescence of its Trp residues. The fluorescent analogue of adenine, 2 aminopurine (2-aPu), introduced in a DNA sequence can be used to observe conformational transitions in a DNA substrate. It was suggested earlier that AP endonucleases have a rigid structure pre-organized for binding of AP sites in DNA (4). In our work, the conformational dynamics of the substrate and Ape1 was investigated by stopped-flow kinetics with fluorescence detection. This approach is well suited to follow the fluorescence changes in a millisecond time range.
The structures of 12-mer oligonucleotide duplexes used as the substrates are presented in Figure 1. The specific substrates contained a natural AP site or its tetrahydrofuran analogue (X). In the non-specific substrate a dG residue was located in the same position as AP or X. To follow conformational transitions in the DNA molecule, the substrates contained a 2 aPu residue either 5? or 3? to AP or X sites.
Figure 1: Structures of DNA substrates used in this work.
The analysis has shown that the enzyme?s affinity for non-specific substrates is two orders of magnitude lower in comparison with that for specific substrates. Moreover, the interaction of Ape1 with uncleavable oligonucleotides can be described only by two reversible stages and doesn?t lead to product formation. In the experiments with 2 aPu-containing substrates, a-Pu, 2-aminopurine
The Trp fluorescence traces obtained under single turnover conditions for different concentrations of specific and non-specific substrates and constant concentration of Ape1 are presented in Figure 2a. For detection of fluorescent traces with substrates containing 2 aPu, the protein concentration was varied, while the substrate concentration remained constant (Figure 2b). Multiple transitions were observed in the fluorescence traces, corresponding to the different stages of this catalytic process. These results are suggestive of conformational mobility both in the active site of Ape1 and in the DNA substrates. The quantitative analysis of the data using DynaFit software produced the minimal kinetic schemes for each substrate and the rate constants of their individual steps.
Figure 2: Typical fluorescence traces observed for DNA cleavage by the Ape1 in the cases of specific substrates (a) with 2 aPu, (b) without 2 aPu.
DNA is incised slower than in the case of the natural duplexes without 2 aPu; this result was also confirmed by electrophoresis with the 32P labelled substrates. The difference observed in the rates can be explained by different affinity of the enzyme?s active site for DNA substrates with 2 aPu and without it (5).
In summary, in this work we have studied kinetic and dynamic features of the mechanism of one of the main human repair enzymes, Ape1. Using the fluorescence stopped-flow technique the conformational mobility of the Ape1 and DNA substrates at the recognition and chemical stages of the catalytic cycle was detected. The observed conformational dynamics suggests the multi-stage character of recognition of damaged sites in DNA and their incision, and can explain the origins of the high specificity of the Ape1 enzyme.
This work was supported by grants from the Wellcome Trust (U.K.) (070244/Z/03/Z), the Siberian Division of the Russian Academy of Sciences (Nos. 51, 60), RFBR (05-04-48447, 05-04-48619, 06-04-49263, 07-04-00191), and from INTAS (04-83-3849).
References and Footnotes
Lyubov Yu. Pshenichnikova1
1Institute of Chemical Biology & Fundamental Medicine