Mammalian L1 retrotransposons as genetic characters

DNA修复诱发突变的机制。我们已经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修复位置两侧的DNA突变，并发现确实如此。特别是，我们引入到基于SV40的Episome中的修复预先形成的正常发生的DNA错对所产生的修复中间产物在低频率但在统计上显著的频率下容易受到APOBEC3(A3)C-脱氨酶介导的错误倾向过程的影响。碱基切除修复(BER)和错配修复通路(MMR)都参与了TPC-偏好A3B脱氨酶必需的单链底物的产生。这一过程产生了类似于各种癌症中典型的突变子表型的突变。突变子表型的机制基础尚不清楚，但我们的研究表明，通常情况下，无错误的DNA修复过程可以作为突变子，提供迄今意想不到的遗传变化来源，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。我们发现这种差异是由于双功能DNA糖基酶NEIL2的表达增加，该酶通过扰动典型的BER使乳腺癌细胞对A3B介导的突变和双链断裂(DSB)敏感。NEIL2通常参与氧化碱基的去除，氧化应激条件下会积累氧化碱基，而氧化应激通常发生在癌细胞中。我们发现，在纯化的BER系统中，NEIL2在基本位置篡夺了典型的BER DNA裂解酶APE1，并且NICK的NEIL2产物不是BER途径中下一种酶-β聚合酶的不良底物。然而，NICK的NEIL2产物可以作为MMR酶Exo1的进入位点，在体外产生单链DNA，这将对A3B和DSB都敏感。我们的研究结果表明，NEIL2或Exo1的缺失可以减轻A3B表达引起的DNA损伤，表明NEIL2的异常表达可以中止正常的BER，并驱动其中间产物进入MMR途径产生A3底物。MMR参与A3介导的突变的体内证据刚刚发表(Mas-Ponte&Supek：DNA错配修复促进APOBEC3介导的人类癌症中的弥漫性过度突变。大自然的吉内特。2020)。我们现在正在使用质谱学来分析氧胁迫条件下NEIL2相互作用的蛋白质。

MECHANISMS OF DNA REPAIR INDUCED MUTAGENESIS. We had implemented an experimental system to determine in vivo whether DNA repair can induce mutations in DNA that flanked the site of DNA repair and found that it does. In particular, the repair intermediates generated from repairing preformed normally occurring DNA mispairs that we introduced into an SV40-based episome were vulnerable at a low but statistically significant frequency to an APOBEC3 (A3) C-deaminase-mediated error-prone process. Both base excision repair (BER) and mismatch repair pathways (MMR) were involved in generating the obligatory single stranded substrate for TpC-preferring A3B deaminase. This process produced mutations similar to those typical of the mutator phenotypes in various cancers. The mechanistic basis of the mutator phenotype is not known but our studies suggested that normally error-free DNA repair processes can act as 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 found that this difference was due to elevated expression of the bifunctional DNA glycosylase, NEIL2, which sensitizes breast cancer cells to both A3B-mediated mutations and double strand breaks (DSBs) by perturbing canonical BER. NEIL2 is normally involved in the removal of oxidized bases, which can accumulate under conditions of oxidative stress that often occurs in cancer cells. We showed that NEIL2 usurps the canonical BER DNA lyase, APE1, at abasic sites in a purified BER system, and that the nicked NEIL2 product is a poor substrate for the next enzyme in the BER pathway, beta polymerase. However, the nicked NEIL2 product can serve as an entry site for the MMR enzyme Exo1 in vitro to generate single-stranded DNA, which would be susceptible to both A3B and DSBs. Our findings that NEIL2 or Exo1 depletion mitigates the DNA damage caused by A3B expression, indicates that aberrant NEIL2 expression can abort normal BER and drive its intermediates into an MMR pathway to generate A3 substrates. In vivo evidence for the involvement of MMR in A3-mediated mutations has just been published (Mas-Ponte & Supek: DNA mismatch repair promotes APOBEC3-mediated diffuse hypermutation in human cancers. Nature Genet. 2020). We are now using mass spectrometry to profile NEIL2-interacting proteins under conditions of oxygen stress.