INTERACTIONS OF DNA POLYMERASE WITH DNA SUBSTRATES

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. C to T and CC to TT transition mutations are the most frequent mutations observed in skin cancer. A deamination hypothesis was proposed to address the generation of those mutations. In this hypothesis, a cytosine or 5-methylcytosine photoproduct deaminates to form a uracil or thymine photoproduct. Those uracil or thymine moieties in the photoproduct form Watson-Crick base-pair preferentially with adenine. After two steps of DNA replication, C to T transition mutation results. To evaluate the importance of deamination in the C to T transition mutation, we took advantage of previous core research and began a collaboration to measure the kinetics of deamination of cytosine and 5-methylcytosine photoproducts. Our approach for the deamination measurement is an extension of the coupled enzymatic digestion MS/MS assay that we already developed in core and published. Because our ion-trap mass spectrometer does not have enough resolving power to separate the deaminate d a nd undeaminated species in normal MS/MS mode, we used the higher resolving power "zoom-scan" MS/MS to follow the deamination process. The "zoom-scan" MS/MS provides accurate measurement of the extent of deamination. The deamination reaction is a pseudo-first-order kinetics, as demonstrated by a straight line while plotting ln(1 - %undeamination) vs. deamination time. We are determining rate constants of deamination of various model oligodeoxynucleotides that are photodamaged, the effect of sequence on the kinetics, and the activation energies and preexponential factors after obtaining rate constants at different temperatures.