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

Area of Science:

• Molecular Biology
• Immunology
• Genetics

Background:

• The activation-induced cytidine deaminase (AID)/apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like (APOBEC) family of enzymes deaminates cytosines in single-stranded DNA (ssDNA).
• These enzymes are crucial for innate and adaptive immunity, mediating viral inactivation and antibody diversification through mutations and strand breaks.
• Evidence suggests APOBEC3A (A3A) and APOBEC3B deaminate cytosines on the lagging-strand template during DNA replication, but direct visualization of the resulting uracils has been lacking.

Purpose of the Study:

• To develop a tool for direct detection of uracil lesions generated by AID/APOBEC enzymes in mammalian cells.
• To investigate the localization and behavior of uracil lesions in relation to DNA replication and APOBEC3A activity.
• To explore the interplay between APOBEC3A, replication protein A (RPA), and replication stress in the context of DNA damage.

Main Methods:

• Engineered a uracil-detecting protein, UdgX, for mammalian expression and immunohistochemistry.
• Utilized immunofluorescence microscopy to visualize UdgX localization in cells expressing APOBEC3A (A3A) and treated with cisplatin.
• Performed co-localization studies with UdgX, A3A, and replication protein A (RPA).
• Confirmed interactions and inhibition using purified proteins in vitro.

Main Results:

• UdgX specifically binds to uracils in ssDNA, showing a preference over uracil in DNA base pairs.
• UdgX localizes to the nucleus and forms distinct foci when co-expressed with A3A or when cells are treated with replication-blocking cisplatin.
• Uracils generated by A3A colocalize with RPA, and RPA inhibits A3A activity on ssDNA, though incompletely under replication stress conditions.

Conclusions:

• The engineered UdgX protein provides direct evidence for uracil formation by A3A in mammalian cells.
• Replication stress, induced by cisplatin, enhances the accumulation of uracil lesions and their association with RPA.
• The findings suggest that despite RPA's inhibitory role, A3A activity during replication stress can lead to the accumulation of characteristic APOBEC signature mutations.