Biochemical Analysis of Hypermutation by the Deoxycytidine Deaminase APOBEC3A

Robin P Love,Huixin Xu,Linda Chelico

doi:10.1074/jbc.m112.393181

Abstract

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 μm). 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.

Highlights

The single-stranded DNA (ssDNA) cytosine deaminase activity of the HIV restriction factor APOBEC3A was analyzed
To determine whether Apo3A has a conservative or more promiscuous target motif, we determined the specific activity of the enzyme on ssDNA substrates (85 nt) containing 5Ј-TTC, 5Ј-ATC, 5Ј-CTC, 5Ј-GTC, or 5Ј-ACC motifs embedded within sequences containing no other cytosines but were otherwise random (Table 1)
There is little biochemical information available for Apo3A to understand the mechanism by which the enzyme catalyzes deaminations and searches for deamination motifs

Summary

Background

The ssDNA cytosine deaminase activity of the HIV restriction factor APOBEC3A was analyzed. 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. Apo enzymes are most commonly associated with their ability to restrict HIV This largely occurs only in the absence of the HIV virus infectivity factor (Vif) protein which facilitates the ubiquitination and degradation of many Apo members (14 –18). During HIV replication, once (Ϫ)DNA synthesis begins from reverse transcription of the HIV RNA genome, the RNase H activity of HIV reverse transcriptase begins degrading the RNA template [24] At this moment ssDNA is uncovered at regions between remaining complementary RNA fragments and is vulnerable to cytosine deamination until the (ϩ)DNA is synthesized [22]. The jumping motions [29] or intersegmental

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION