Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Kinetics of the Initial Step of Base Excision Repair in Trinucleotide Repeats
Dynamic mutations arising from trinucleotide repeat expansion cause a number of grave hereditary neurodegenerative diseases and possibly contribute to neurodegeneration during normal aging. In the case of terminally differentiated neurons such expansion can be caused by DNA base excision repair (BER). This hypothesis was confirmed by experiments in which expansion of CAG triplets characteristic of Huntington disease was initiated in the course of normal repair of the damaged base 8-oxoguanine (8-oxoG) (1). Yet it is unclear how such a DNA base lesion and its repair might cause the expansion. Besides, there is extremely little information is available on efficiency of BER enzyme and their substrate specificity when DNA substrate contains trinucleotide repeats. Thus it would be very useful to gain kinetic parameters of reaction involving DNA glycosylase, the main participants of BER, and such DNA substrates. First of all, we were interested in how the position of 8-oxoG in a substrate containing a run of CAG repeats can influence on its excision rate by human 8-oxoguanine glycosylase (OGG1). For this purpose we have determined rate constants of 8-oxoG excision (k2) by OGG1 enzyme from CAG-substrate depending on the position of the damaged triplet. Also for these substrates we have determined the rate constants of DNA product release from a complex with OGG1 (k3). We have then compared the rates of 8-oxoG between purified enzyme and nuclear extract of the human hepatoma cell line (LICH). We have observed that k2 of purified OGG1 and the rate of excision in the extracts exhibited a similar dependence on the position of the lesion in the substrate. Therefore we suggest that the reaction rate in the cell extract is limited not by the product release stage, as in the case of pure protein, but by the catalytic step of reaction, as shown previously for the activity of OGG1 in the presence of AP-endonuclease APEX1 (2). In the case of substrates containing 8-oxoG in CGG repeats, typical for fragile X syndrome, the kinetic parameters were comparable with those for the CAG-substrates. Given that potentially not only repair of 8-oxoG can lead to expansion of triplets, we have obtained the values of Michaelis constant and catalytic constants for the reaction of uracil excision by human uracil-DNA glycosylase (UNG) from CAG-run substrates containing a single uracil, the product of cytosine deamination. Remarkably, despite the strict dependence of both constants on the position of the damaged triplet in the substrate, the specificity constant (kcat/KM) did not significantly change.
Figure. 1. Dependence of the excision rate constant (k2) of oxoG from CAG substrates by purified OGG1 (black bars) and the excision rate (v) of oxoG in LICH extracts (gray bars) on the position of oxoG in the substrate containing seven CAG trinucleotides. The number in the name of the substrate corresponds to the damaged trinucleotide.
This work was supported in part by Russian Foundation for Basic Research (10-04-91058-PICS_a)
SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia