APOBEC3A Loop 1 Is a Determinant for Single-Stranded DNA Binding and Deamination

Abstract

The apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) family of proteins functions in the innate immune system. The A3 proteins are interferon inducible and hypermutate deoxycytidine to deoxyuridine in foreign single-stranded DNA (ssDNA). However, this deaminase activity cannot discriminate between foreign and host ssDNA at the biochemical level, which presents a significant danger when A3 proteins gain access to the nucleus. Interestingly, this A3 capability can be harnessed when coupled with novel CRISPR-Cas9 proteins to create a targeted base editor. Specifically, A3A has been used in vitro to revert mutations associated with disease states. Recent structural studies have shown the importance of loop regions of A3A and A3G in ssDNA recognition and positioning for deamination. In this work, we further examined loop 1 of A3A to determine how it affects substrate selection, as well as efficiency of deamination, in the hopes of advancing the potential of A3A in base editing technology. We found that mutating residue H29 enhanced deamination activity without changing substrate specificity. Also interestingly, we found that increasing the length of loop 1 decreases substrate specificity. Overall, these results lead to a better understanding of substrate recognition and deamination by A3A and the A3 family of proteins.

Loop 1 of APOBEC3C Regulates its Antiviral Activity against HIV-1

“…A similar model was discussed by Solomon and coworkers, which demonstrated that loop 1 residues of hA3G-CTD-2K3A-E259A (a catalytically inactive form of A3G-CTD) strongly interact with substrate ssDNA and that this distinguishes catalytic binding from non-catalytic binding [81]. Interestingly, loop 1 of A3A was found to be important for substrate specificity but not for substrate binding affinity [82], while loop 1 of A3H especially residue R26, plays a triple role for RNA binding, DNA substrate recognition, and catalytic activity likely by positioning the DNA substrate in the active site for effective catalysis [83]. In accordance with this, our study claims that 25 RK 26 substitution in loop 1 of A3C provides the microenvironment that drives the flexibility in substrate binding and enzymatic activity.…”

Loop 1 of APOBEC3C regulates its antiviral activity against HIV-1

“…A similar model was discussed by Solomon and coworkers, which demonstrated that loop 1 residues of hA3G-CTD-2K3A-E259A (a catalytically inactive form of A3G-CTD) strongly interact with substrate ssDNA and that this distinguishes catalytic binding from non-catalytic binding [81]. Interestingly, loop 1 of A3A was found to be important for substrate specificity but not for substrate binding affinity [82], while loop 1 of A3H especially residue R26, plays a triple role for RNA binding, DNA substrate recognition, and catalytic activity likely by positioning the DNA substrate in the active site for effective catalysis [83]. In accordance with this, our study claims that 25 Furthermore, the increased mobility of DNA binding regions carrying the substitutions in C2 and hA3C-S61P.S188I, respectively, compared to hA3C (Suppl.…”

Human activation-induced deaminase lacks strong replicative strand bias or preference for cytosines in hairpin loops

“…Recent studies have suggested that loop 1 of A3A, which is shortest among all the AID/APOBEC enzymes, plays a key role in its high activity by making its active site more open ( 59 , 60 ). We wondered whether the presence of a larger loop 1 sequence in AID ( Supplementary Figure S5A ) could explain the enzyme's lack of preference for the more structured hairpin loop sequences.…”