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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 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.

SUBMITTER: Stewart JA

PROVIDER: S-EPMC7672425 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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

Stewart Jessica A JA Schauer Grant G Bhagwat Ashok S AS

Nucleic acids research 20201101 20

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 covalen ...[more]

PMID: 33074285

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Project description:Expansive growth of neural progenitor cells (NPCs) is a prerequisite to the temporal waves of neuronal differentiation that generate the six-layered neocortex, while also placing a heavy burden on proteins that regulate chromatin packaging and genome integrity. This problem is further reflected by the growing number of developmental disorders caused by mutations in chromatin regulators. ATRX gene mutations cause a severe intellectual disability disorder (α-thalassemia mental retardation X-linked (ATRX) syndrome; OMIM no. 301040), characterized by microcephaly, urogenital abnormalities and α-thalassemia. Although the ATRX protein is required for the maintenance of repetitive DNA within heterochromatin, how this translates to disease pathogenesis remain poorly understood and was a focus of this study. We demonstrate that Atrx(FoxG1Cre) forebrain-specific conditional knockout mice display poly(ADP-ribose) polymerase-1 (Parp-1) hyperactivation during neurogenesis and generate fewer late-born Cux1- and Brn2-positive neurons that accounts for the reduced cortical size. Moreover, DNA damage, induced Parp-1 and Atm activation is elevated in progenitor cells and contributes to their increased level of cell death. ATRX-null HeLa cells are similarly sensitive to hydroxyurea-induced replication stress, accumulate DNA damage and proliferate poorly. Impaired BRCA1-RAD51 colocalization and PARP-1 hyperactivation indicated that stalled replication forks are not efficiently protected. DNA fiber assays confirmed that MRE11 degradation of stalled replication forks was rampant in the absence of ATRX or DAXX. Indeed, fork degradation in ATRX-null cells could be attenuated by treatment with the MRE11 inhibitor mirin, or exacerbated by inhibiting PARP-1 activity. Taken together, these results suggest that ATRX is required to limit replication stress during cellular proliferation, whereas upregulation of PARP-1 activity functions as a compensatory mechanism to protect stalled forks, limiting genomic damage, and facilitating late-born neuron production.

Dataset Information

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

Publications

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

Similar Datasets

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