Methylcytosine and normal cytosine deamination by the foreign DNA restriction enzyme APOBEC3A

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• DNA methylation is crucial for gene regulation.
• TET oxidases and APOBEC deaminases can modify DNA bases.
• The specific roles of APOBEC deaminases in DNA base editing are still being elucidated.

Purpose of the Study:

• To investigate the DNA editing capabilities of human APOBEC3A (A3A) and APOBEC3G (A3G).
• To determine the activity of A3A and A3G on 5-methylcytosine (MeC) and normal cytosine (C) in DNA.
• To explore the cellular relevance of A3A-mediated DNA editing.

Main Methods:

• In vitro biochemical assays using single-stranded DNA substrates.
• Cellular assays involving interferon-induced expression of endogenous A3A.
• Knockdown experiments to identify the source of cellular DNA deaminase activity.
• Analysis of DNA methylation status in chromosomal and transfected DNA.

Main Results:

• Human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and C to uracil (U) in ssDNA.
• APOBEC3G (A3G) shows significantly lower activity for both substrates compared to A3A.
• Endogenous A3A, induced by interferon, edits MeC and C in transfected plasmid DNA but not bulk chromosomal DNA.
• Knockdown experiments confirm endogenous A3A as the source of cellular DNA deaminase activity.

Conclusions:

• This study provides the first evidence of non-chromosomal DNA MeC to T editing in human cells, mediated by A3A.
• The broad substrate specificity of A3A suggests an evolutionary adaptation for innate immune functions, particularly foreign DNA clearance.
• A3A's editing activity is primarily observed on extrachromosomal DNA, highlighting potential roles in host defense mechanisms.