Biochemical Analysis of Hypermutation by the Deoxycytidine Deaminase APOBEC3A

被引:71
作者
Love, Robin P. [1 ]
Xu, Huixin [1 ]
Chelico, Linda [1 ]
机构
[1] Univ Saskatchewan, Dept Microbiol & Immunol, Saskatoon, SK S7N 5E5, Canada
基金
美国国家卫生研究院; 加拿大自然科学与工程研究理事会;
关键词
SINGLE-STRANDED-DNA; HUMAN-IMMUNODEFICIENCY-VIRUS; INDUCED CYTIDINE DEAMINASE; SOMATIC HYPERMUTATION; VIF PROTEIN; HIV-1; VIF; REVERSE-TRANSCRIPTASE; ANTIVIRAL ACTIVITY; ENZYME APOBEC3G; HUMAN-CELLS;
D O I
10.1074/jbc.M112.393181
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
APOBEC3A belongs to a family of single-stranded DNA (ssDNA) DNA cytosine deaminases that are known for restriction of HIV through deamination-induced mutational inactivation, e. g. APOBEC3G, or initiation of somatic hypermutation and class switch recombination (activation-induced cytidine deaminase). APOBEC3A, which is localized to both the cytoplasm and nucleus, not only restricts HIV but can also initiate catabolism of cellular DNA. Despite being ascribed these roles, there is a paucity of data available on the biochemical mechanism by which APOBEC3A deaminates ssDNA. Here we assessed APOBEC3A deamination activity on ssDNA and in dynamic systems modeling HIV replication (cytoplasmic event) and DNA transcription (nuclear event). We find that APOBEC3A, unlike the highly processive APOBEC3G, exhibits low or no processivity when deaminating synthetic ssDNA substrates with two cytosines located 5-63 nucleotides apart, likely because of an apparent K-d in the micromolar range (9.1 mu M). APOBEC3A was able to deaminate nascently synthesized (-) DNA in an in vitro model HIV replication assay but induced fewer mutations overall in comparison to APOBEC3G. However, the data indicate that the target deamination motif (5'-TC for APOBEC3A and 5'-CC for APOBEC3G) and not the number of mutations best predicted the ability to mutationally inactivate HIV. We further assessed APOBEC3A for the ability to deaminate dsDNA undergoing transcription, which could allow for collateral deaminations to occur in genomic DNA similar to the action of activation-induced cytidine deaminase. That APOBEC3A was able to deaminate dsDNA undergoing transcription suggests a genomic cost of a deamination-based retroviral restriction system.
引用
收藏
页码:30812 / 30822
页数:11
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