Multiple mutations are required for cancer development, and genome
sequencing has revealed that several cancers, including breast, have somatic
mutation spectra dominated by C-to-T transitions1–9.
Most of these mutations occur at hydrolytically disfavored10 non-methylated cytosines
throughout the genome, and are sometimes clustered8. Here, we show that the DNA cytosine deaminase
APOBEC3B (A3B) is a likely source of these mutations. A3B mRNA
is up-regulated in the majority of primary breast tumors and breast cancer cell
lines. Tumors that express high levels of A3B have twice as
many mutations as those that express low levels and are more likely to have
mutations in TP53. Endogenous A3B protein is predominantly
nuclear and the only detectable source of DNA C-to-U editing activity in breast
cancer cell line extracts. Knockdown experiments show that endogenous A3B
correlates with elevated levels of genomic uracil, increased mutation
frequencies, and C-to-T transitions. Furthermore, induced A3B over-expression
causes cell cycle deviations, cell death, DNA fragmentation, γ-H2AX
accumulation, and C-to-T mutations. Our data suggest a model in which
A3B-catalyzed deamination provides a chronic source of DNA damage in breast
cancers that could select TP53 inactivation and explain how
some tumors evolve rapidly and manifest heterogeneity.
Background: APOBEC3A is a myeloid-specific interferon-inducible DNA C to U deaminase implicated in innate immunity. Results: APOBEC3A also elicits MeC to T editing activity in vitro with deoxy-oligonucleotides and in vivo with transfected plasmids. Conclusion: APOBEC3A accommodates both normal and larger DNA cytosine substrates. Significance: The developmental specialization and broader substrate range of APOBEC3A may be an evolutionary adaptation for physiological function in foreign DNA restriction.
APOBEC3G (A3G) is an antiviral protein that binds RNA and single-stranded DNA (ssDNA). The oligomerization state of A3G is likely to be influenced by these nucleic acid interactions. We applied the power of nanoimaging atomic force microscopy technology to characterize the role of ssDNA in A3G oligomerization. We used recombinant human A3G prepared from HEK-293 cells and specially designed DNA substrates that enable free A3G to be distinguished unambiguously from DNA-bound protein complexes. This DNA substrate can be likened to a molecular ruler because it consists of a 235-bp double-stranded DNA visual tag spliced to a 69-nucleotide ssDNA substrate. This hybrid substrate enabled us to use volume measurements to determine A3G stoichiometry in both free and ssDNA-bound states. We observed that free A3G is primarily monomeric, whereas ssDNA-complexed A3G is mostly dimeric. A3G stoichiometry increased slightly with the addition of Mg2+, but dimers still predominated when Mg2+ was depleted. A His-248/His-250 Zn2+-mediated intermolecular bridge was observed in a catalytic domain crystal structure (Protein Data Bank code 3IR2); however, atomic force microscopy analyses showed that the stoichiometry of the A3G-ssDNA complexes changed insignificantly when these residues were mutated to Ala. We conclude that A3G exchanges between oligomeric forms in solution with monomers predominating and that this equilibrium shifts toward dimerization upon binding ssDNA.
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