Delineating a New Mechanism of Foreign DNA Restriction in Human Cells

APOBEC3 DNA胞嘧啶脱氨酶是哺乳动物先天免疫反应的基本组成部分。特别是，APOBEC3A介导人类细胞中的外源DNA限制，其结果类似于限制酶在细菌中的保护作用。干扰素、CpG低聚物、转染双链DNA和一些DNA病毒显著上调该酶的表达水平。从表达apobec3a的细胞中提取的外源DNA含有大量的胞嘧啶脱胺，多达三分之二的胞嘧啶转化为尿嘧啶（检测为C/G到T/A的过渡突变）。首先，我们将使用模型DNA底物和生化方法来验证APOBEC3A倾向于双链DNA底物的假设，而不是识别单链DNA的其他家族成员。其次，亚细胞定位和生化分离实验将用于验证染色体DNA不被APOBEC3A靶向的假设，因为催化活性酶只定位于细胞质或细胞质亚室（如核内体）。最后，我们建议推进我们的新小分子的初步研究，以确定那些特异性抑制APOBEC3A在人类细胞中的活性。我们预计，真正的APOBEC3A抑制剂将使人类细胞更易于基因工程修改。总的来说，我们期望阐明更多apobec3a介导的外源DNA限制机制，并发现先导化合物来暂时中和这种先天防御，使细胞更容易受到基因工程的影响。

The APOBEC3 DNA cytosine deaminases are a fundamental part of the mammalian innate immune response. In particular, APOBEC3A mediates foreign DNA restriction in human cells with an outcome analogous to the protective effects of restriction enzymes in bacteria. This enzyme's expression levels are dramatically up-regulated by interferon, CpG oligos, transfected double-stranded DNA, and some DNA viruses. Foreign DNA recovered from APOBEC3A-expressing cells contains enormous levels of cytosine deamination with as many as two thirds of the cytosines converted to uracils (detected as C/G to T/A transition mutations). First, we will use model DNA substrates and biochemical approaches to test the hypothesis that APOBEC3A prefers double-stranded DNA substrates, as opposed to other family members that recognize single-stranded DNA. Second, subcellular localization and biochemical fractionation experiments will be used to test the hypothesis that chromosomal DNA is not targeted by APOBEC3A because catalytically active enzyme is localized exclusively to the cytoplasm or a cytoplasmic sub-compartment such as the endosomes. Finally, we propose to advance our preliminary studies on novel small molecules to identify those that specifically inhibit APOBEC3A activity in human cells. We anticipate that bona fide APOBEC3A inhibitors will make human cells more amendable to genetic engineering. Overall, we anticipate elucidating more of the APOBEC3A-mediated foreign DNA restriction mechanism and discovering lead compounds to transiently neutralize this innate defense and render cells more susceptible to genetic engineering.