More than half of all SHMs occur around a DNA motif known as the RGYW motif (where R denotes A or G, Y denotes C or T, and W denotes A or T), leading to the suggestion that unknown factors might target AID to these sites. To identify these putative cofactors Chaudhuri et al. used an in vitro assay for AID activity. This assay relied on the observation that although AID purified from B cells can by itself deaminate ssDNA templates, it cannot by itself deaminate an actively transcribed double-stranded DNA SHM substrate (that is, a substrate containing multiple RGYW motifs that cannot form R loops — structures generated during transcription that displace the non-template strand as ssDNA). The ability of protein fractions isolated from B cells to facilitate AID deamination of the SHM substrate led to the identification of a heat-labile complementing factor (CF) of 120–170 kDa.
In further experiments, a 30–35 kDa protein that was highly enriched in fractions containing CF activity was shown to associate with AID. Subsequent analysis identified this as the 32 kDa subunit of RPA (RPA32). RPA is heterotrimeric — consisting of a 70 kDa (RPA70) and a 14 kDa (RPA14) subunit, in addition to RPA32 — and has previously been implicated in DNA replication, recombination and repair. Confirmation of RPA as the CF was provided by the observation that recombinant human RPA containing all three subunits could replace CF in facilitating AID deamination of the actively transcribed SHM substrate. As well as complexes that contain AID, RPA and RGYW-containing DNA substrates, smaller complexes that contain only RPA and the RGYW-containing substrate were observed. Because these smaller complexes were not detected when the deamination reaction was inhibited, the authors suggest that AID-mediated deamination leads to the release of AID from the AID–RPA–DNA complex and that the DNA-bound RPA might enable the recruitment of DNA-repair proteins.
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