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.
Foreign DNA poses an intrinsic threat to cells and the human APOBEC3 proteins prevent its uptake. A molecular understanding of this process will have implications for combating human disease through enhancing innate immune responses and, in some instances, through diminishing the innate responses to enhance the uptake of therapeutic DNA.
|Rathore, Anurag; Carpenter, Michael A; Demir, Özlem et al. (2013) The local dinucleotide preference of APOBEC3G can be altered from 5'-CC to 5'-TC by a single amino acid substitution. J Mol Biol 425:4442-54|
|Land, Allison M; Law, Emily K; Carpenter, Michael A et al. (2013) Endogenous APOBEC3A DNA cytosine deaminase is cytoplasmic and nongenotoxic. J Biol Chem 288:17253-60|
|Leonard, Brandon; Hart, Steven N; Burns, Michael B et al. (2013) APOBEC3B upregulation and genomic mutation patterns in serous ovarian carcinoma. Cancer Res 73:7222-31|
|Burns, Michael B; Lackey, Lela; Carpenter, Michael A et al. (2013) APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 494:366-70|
|Carpenter, Michael A; Li, Ming; Rathore, Anurag et al. (2012) Methylcytosine and normal cytosine deamination by the foreign DNA restriction enzyme APOBEC3A. J Biol Chem 287:34801-8|