Mammalian L1 retrotransposons as genetic characters

DNA修复诱发突变的机制。我们实施了一个实验系统来在体内确定DNA修复是否可以诱导DNA侧翼的突变，并发现确实可以。特别是，在基于SV40的Episome上修复预先形成的正常发生的DNA错配所产生的修复中间产物在低频率但在统计学上显著地容易受到APOBEC介导的易于出错的过程的影响。SiRNA敲除表明，突变需要碱基切除修复和错配修复途径的组件，或可以与这些途径相互作用的因子(例如，增殖细胞核抗原和ATR)，以及TPC偏好的APOBEC胞苷脱氨酶，特别是A3B，它产生的突变类似于各种癌症中典型的突变子表型。因此，正常情况下，无错误的DNA修复过程可以转变为突变，提供迄今意想不到的基因变化来源，这些变化是疾病、衰老和进化变化的基础。我们将我们的Episome突变传感器应用于含有相似A3B水平的已建立的乳腺癌细胞系对，发现其中一些对在突变修复方面存在显著差异。我们分析了这些配对细胞系中的DNA修复酶和相关因素，发现修复诱导突变的细胞和没有修复诱导突变的细胞之间的修复酶和相关因子有很大不同。我们现在正在系统地评估修复酶差异对突变效应的贡献或缺乏，并将用配对细胞系的WGA来补充这些研究，以确定它们是否具有与我们的外体报告获得的一致的突变特征。配对细胞系之间的比较还揭示了除了DNA修复过程中产生的那些底物之外，还有其他几种可能的A3B底物的新来源。我们已经开发了体外系统来证实这些假设，并确定涉及的生化机制。我们还在修改我们编写的Perl程序，该程序用于在上述基于表体的数据集中检测突变特征，以重新检查直系黑猩猩/人类L1化石的大型数据集，以确定可能作为CG含量的函数在过去80Myr灵长类血统中发生的中性进化的突变过程的种类。

MECHANISMS OF DNA REPAIR INDUCED MUTAGENESIS. We implemented an experimental system to determine in vivo whether DNA repair can induce mutations in flanking DNA and found that it does. In particular, the repair intermediates generated from repairing preformed normally occurring DNA mispairs on an SV40-based episome were vulnerable at a low but statistically significant frequency to an APOBEC-mediated error-prone process. SiRNA knockdowns showed that components of both the base excision repair and mismatch repair pathways, or factors that can interact with these pathways (e.g., PCNA and ATR), and TpC-preferring APOBEC cytidine deaminases, particularly A3B, are required for mutagenesis, which produces mutations similar to those typical of the mutator phenotypes in various cancers. Thus, normally error-free DNA repair processes can be turned into mutators providing a heretofore unexpected source of genetic changes that underlie disease, aging and evolutionary change. We applied our episome mutation sensor to pairs of established breast cancer cell lines that contain similar A3B levels and found that some pairs differed dramatically in mutagenic repair. We profiled the DNA repair enzymes and related factors in these paired cell lines and found that they differ dramatically between cells that exhibit repair-induced mutations and those that do not. We are now systematically evaluating the repair enzyme differences for their contribution to the mutagenic effect or lack thereof and will supplement these studies with WGA of the paired cell lines to determine if they harbor mutational signatures consistent with those obtained with our episomal reporter. Comparisons between the paired cell lines also revealed several other possible novel sources of A3B substrates in addition to those generated during DNA repair. We have developed in vitro systems to corroborate these suppositions and determine the biochemical mechanisms involved. We are also modifying our Perl program that we had written to detect mutational signatures in the above episomal based data sets to re-examine a large dataset of orthologous chimpanzee/human L1 fossils to determine the kinds of mutational processes that could underlie the neutral evolution that occurred in the primate lineage over the past 80 Myr as a function of CG content.