INTERACTIONS OF DNA POLYMERASE WITH DNA SUBSTRATES

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。C到T和CC到TT转换突变是在皮肤癌中观察到的最常见的突变。提出了脱氨基假说来解决这些突变的产生。在这个假设中，胞嘧啶或5-甲基胞嘧啶光产物脱氨基形成尿嘧啶或胸腺嘧啶光产物。光产物中的那些尿嘧啶或胸腺嘧啶部分优先与腺嘌呤形成沃森-克里克碱基对。经过两个步骤的DNA复制，C到T转换突变的结果。为了评估脱氨基在C到T转换突变中的重要性，我们利用以前的核心研究，开始合作测量胞嘧啶和5-甲基胞嘧啶光产物脱氨基的动力学。我们的脱氨基测量方法是我们已经在核心开发并发表的偶联酶消化MS/MS测定的延伸。由于我们的离子阱质谱仪在正常MS/MS模式下没有足够的分辨率来分离脱氨基和未脱氨基的物质，我们使用更高分辨率的“变焦扫描”MS/MS来跟踪脱氨基过程。“放大扫描”MS/MS提供脱氨基程度的准确测量。脱氨基反应是假一级动力学，如在绘制ln（1 - %脱氨基）对脱氨基时间时的直线所示。我们正在确定脱氨基的各种模型的寡脱氧核苷酸是光损伤，序列的动力学的影响，并在不同温度下获得速率常数后的活化能和指前因子的速率常数。

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.