Модулирование генопротекторных механизмов в клетках человека с использованием аналога НАДН

Секция 1. Молекулярные механизмы генетических процессо

Молекулярный механизм Гипермутабильности V3 региона поверхностного гликопротеина gp120 ВИЧ1

Секция 1. Молекулярные механизмы генетических процессо

Oxidative sensitivity of alternative functions of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase

The glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is susceptible to inflammatory oxidants such as hypochlorite (OCl ) and hydrogen peroxide (H2O2). Evidence [1] suggests that the monomeric form of GAPDH exhibits uracil-DNA glycosylase (UDG) activity. As oxidative damage has often been implicated as a possible cause of carcinogenesis, the sensitivity of UDG to inflammatory oxidants was investigated. An assay, modified from Sirover (1979) [2], was developed which successfully quantitated the UDG activity of both bacterial UDG and UDG extracted from a human cell line, HCT 116. UDG activity in these cells was proportional to cell number, with an upper limit of 2.5 x 10^ cells, possessing 3033U UDG/mg protein. Aliquots containing 2.5 x 10^ cells were exposed to the inflammatory oxidants, OCl and H2O2. Exposure of HCT 116 cells to OCl up to a concentration of 5 x lO\u27^M resulted in no change in UDG activity released from these cells. However, exposure to OCl at concentrations higher than 5 x lO\u27^M resulted in complete loss of cellular UDG activity. In contrast, UDG activity was not inhibited at all in HCT 116 cells exposed to H2O2 up to a concentration of lO\u27^M. Bacterial UDG, however, was not inhibited by either OCl\u27 up to a concentration of lO\u27^M, or H2O2 up to a concentration of lO\u27^M. As bacterial UDG displays different properties to human UDG, it may not be used as a model to investigate the mode of inactivation of human UDG by OCl

Effect of DNA Base Modification on Polymerase Chain Reaction Efficiency and Fidelity

Polymerase stop assays, used to quantify DNA damage, assume single lesions are sufficient to block thermostable DNA polymerase progression. To explore this assumption, 90 base oligonucleotides containing normal or modified DNA bases were amplified using real-time PCR. Data implied that the PCR efficiency was influenced to differing degrees depending on which base lesion was present on the input oligonucleotide; specifically, while reactions with templates containing a single 8-oxo-7,8-dihydro-2í-deoxyguanosine (8-oxodG) were not noticeably altered, the presence of a single 8-oxo-7,8-dihydro-2í-deoxyadenosine, an abasic site, or a cis-syn thymidine dimer (TT dimer) dramatically delayed amplification. In addition, the presence of two tandem 8-oxodGs substantially hindered amplification when compared with two 8-oxodGs separated by 13 bases which indicated that the position of lesions also influenced the PCR. To quantify variations in amplification, novel mathematical formulae were developed which report differences in exponential amplification as rates of damage bypass. These treatments assume each template in the PCR is damaged to the same degree. Quantification of damage to cellular DNA, which is a mixture of damaged and undamaged template, required further refinement of real-time PCR mathematics; differences in amplification were defined in terms of damage probability (lesion frequency) rather than lesion bypass rate. The validity of these formulae was determined using DNA samples quantified previously using current polymerase stop methods. In addition to impacting reaction efficiency, DNA base modifications decreased reaction fidelity. In reactions with templates containing 8-oxodGs, both the normal Watson/Crick association with dCMP as well as the incorporation of dAMP occurred at the lesion site. Despite similar structural characteristics, the existence of 8-oxodA resulted in a pronounced n-1 deletion in addition to the normal association with dTMP. Sequence data from abasic and TT dimer modifications were inconclusive but suggested the presence of multiple nucleotide incorporation events opposite the modifications. The present work enabled the adaptation of real-time PCR for DNA damage quantification, identified DNA base lesions as potential PCR mutagens, and provides the basis for further refinement of polymerase stop assays as research and clinical tools to monitor DNA damage and repair