Journal: bioRxiv

Article Title: Molecular mechanism for regulating APOBEC3G DNA editing function by the non-catalytic domain

doi: 10.1101/2024.03.11.584510

Figure Lengend Snippet: AA-facilitated DNA editing in hairpin forming sequences performed with the hyperactive variant rA3G R8/P247K/Q317K . (a) Comparative modeling of rA3G bound to AA-DNA (this study), the editing motif CCC-DNA (in pink, modeled from PDB 6BUX ) and a DNA hairpin structure with a tetraloop and a 12-bp stem length (in green, based on PDB 8FIK ). A potential connection path (∼46 Å) between the hairpin DNA (with a 10-bp stem length) and the AA motif is indicated by a black dotted line and labeled as ‘portion of a 3’overhang’. (b) Location of the AA motif on 3’ overhang affects editing efficiency. Deamination activity was monitored on hairpin DNA 1 substrates carrying a 10-bp hairpin stem, a tetraloop CCCC, and a 3’overhang. A single AA motif was placed at various locations on the 3’ overhang. A negative control with 5’-TTTT in the hairpin loop and a single A 23 A 24 motif in the 3’overhang was included. The results show that the top three edited hairpin DNA with the stem length of 10 bp are those carrying a single AA motif A 20 A 21 , A 23 A 24 , or A 26 A 27 . (c) Effect of stem length on editing efficiency. Deamination activity was monitored on hairpin DNA 2 substrates with varying stem lengths from 4 bp to 12 bp. The position of the AA motif was kept the same in all substrates as the position of A 23 A 24 in the 10-bp stem loop DNA substrate. Three linear DNA substrates were included as linear DNA controls. The results show that hairpin DNA with short stem lengths (4 bp or 5 bp) are poorly edited, whereas hairpin DNA with long stem lengths (10 bp or 11 bp) are well edited.

Article Snippet: Reactions (20 μl) containing the purified protein (rA3G R8 or indicated mutants, with specified concentrations) and individual DNA substrates (with indicated sequences and concentrations) were incubated at 37°C for an indicated duration in DNA deamination buffer [25 mM HEPES at pH 7, 250 mM NaCl, 1 mM DTT, 0.1% Triton X-100, 0.1 mg/ml recombinant albumin (New England Biolabs), and 0.1 mg/ml RNase A (QIAGEN)].

Techniques: Variant Assay, Labeling, Activity Assay, Negative Control