Visualization of uracils created by APOBEC3A using UdgX shows colocalization with RPA at stalled replication forks

Abstract The AID/APOBEC enzymes deaminate cytosines in single-stranded DNA (ssDNA) and play key roles in innate and adaptive immunity. The resulting uracils cause mutations and strand breaks that inactivate viruses and diversify antibody repertoire. Mutational evidence suggests that two members of t...

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Bibliographic Details
Published in:Nucleic acids research 2020-11, Vol.48 (20), p.e118-e118
Main Authors: Stewart, Jessica A, Schauer, Grant, Bhagwat, Ashok S
Format: Article
Language:English
Subjects:
Cell Nucleus - metabolism
Cisplatin - pharmacology
Cross-Linking Reagents - pharmacology
Cytidine Deaminase - genetics
Cytidine Deaminase - metabolism
Cytosine - metabolism
DNA - chemistry
DNA - metabolism
DNA Replication
DNA, Single-Stranded - metabolism
HEK293 Cells
HeLa Cells
Humans
Imidazoles - pharmacology
Methods Online
Protein Engineering
Proteins - genetics
Proteins - metabolism
Replication Protein A - metabolism
Uracil - analysis
Uracil - chemistry
Uracil - metabolism
Uracil-DNA Glycosidase - genetics
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Summary:Abstract The AID/APOBEC enzymes deaminate cytosines in single-stranded DNA (ssDNA) and play key roles in innate and adaptive immunity. The resulting uracils cause mutations and strand breaks that inactivate viruses and diversify antibody repertoire. Mutational evidence suggests that two members of this family, APOBEC3A (A3A) and APOBEC3B, deaminate cytosines in the lagging-strand template during replication. To obtain direct evidence for the presence of these uracils, we engineered a protein that covalently links to DNA at uracils, UdgX, for mammalian expression and immunohistochemistry. We show that UdgX strongly prefers uracils in ssDNA over those in U•G or U:A pairs, and localizes to nuclei in a dispersed form. When A3A is expressed in these cells, UdgX tends to form foci. The treatment of cells with cisplatin, which blocks replication, causes a significant increase in UdgX foci. Furthermore, this protein- and hence the uracils created by A3A- colocalize with replication protein A (RPA), but not with A3A. Using purified proteins, we confirm that RPA inhibits A3A by binding ssDNA, but despite its overexpression following cisplatin treatment, RPA is unable to fully protect ssDNA created by cisplatin adducts. This suggests that cisplatin treatment of cells expressing APOBEC3A should cause accumulation of APOBEC signature mutations.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkaa845