INTERACTIONS OF DNA POLYMERASE WITH DNA SUBSTRATES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在皮肤癌中观察到的最常见的突变是C到T和CC到TT转换突变。一种脱氨基假说被提出来解决这些突变的产生。在这个假说中，胞嘧啶或5-甲基胞嘧啶的光产物脱氨基，形成尿嘧啶或胸腺嘧啶的光产物。光产物中的尿嘧啶或胸腺嘧啶部分优先与腺嘌呤形成Watson-Crick碱基对。在DNA复制的两个步骤后，C到T的转变突变。为了评估脱氨基在C到T转变突变中的重要性，我们利用以前的核心研究，开始了一项合作，以测量胞嘧啶和5-甲基胞嘧啶光产物的脱氨基动力学。我们的脱氨基测定方法是我们已经在CORE中开发并发表的耦合酶消化MS/MS分析的扩展。由于我们的离子陷阱质谱仪在正常的MS/MS模式下没有足够的分辨率来分离脱胺和未脱胺物种，所以我们使用了分辨率较高的“变焦扫描”MS/MS来跟踪脱胺过程。“变焦扫描”MS/MS可准确测量脱氨的程度。脱胺反应为假一级反应，ln(1-%脱氨率)与脱胺时间呈直线关系。我们正在测定各种光损伤模型寡核苷酸的脱氨速率常数，序列对动力学的影响，以及在不同温度下得到的速率常数后的活化能和指前因子。

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.