Single-stranded DNA Scanning and Deamination by APOBEC3G Cytidine Deaminase at Single Molecule Resolution

Abstract

Background: Apo3G, an ssDNA-dependent C deaminase, inactivates HIV-1 in T cells by C to T hypermutation. Results: smFRET is used to detect Apo3G scanning and C-deamination on ssDNA. Conclusion: Apo3G scans ssDNA randomly and bidirectionally, favoring nonrandom 3Ј to 5Ј deamination. Significance: This smFRET study describes a broadly applicable approach to visualize motion and catalysis in real time by an enzyme that scans ssDNA.

A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer

“…alyzed by the same enzyme during a single APOBEC-ssDNA binding event. Processive catalysis by APOBECs has been shown biochemically to generate single along with short and long clusters of mutations for AID, Apo3G (36,38,61), and in this study for Apo3A.…”

Suppression of HIV-1 Infection by APOBEC3 Proteins in Primary Human CD4 + T Cells Is Associated with Inhibition of Processive Reverse Transcription as Well as Excessive Cytidine Deamination

“…In light of the lack of obviously preferred sites of pausing or termination and the calculated approximately seven A3G molecules present per virion (71), this model seemingly requires that APOBEC3 proteins bind throughout the length of the gRNA, as well as translocate relatively freely from one binding site to another. Indeed, in support of this idea, this behavior has been documented for the binding of recombinant A3G to single-stranded DNA in vitro (65,72,73). Planned future studies that map APOBEC3 protein binding sites on viral RNA will help address the relationship between RNA interactions and effects on reverse transcription.…”

Biochemical Analysis of Hypermutation by the Deoxycytidine Deaminase APOBEC3A

“…In this type of processive movement, the enzyme can slide and make microscopic dissociations and reassociations with the DNA (termed hopping, jumping, or intersegmental transfer), without diffusing into the bulk solution to efficiently search for target sequences where catalysis takes place (25,28,29). Apo3G has also been shown to prefer deamination toward the 5Ј-end of linear ssDNA due to a catalytic orientation specificity (27,62,63). It is not known if these hallmark features of Apo3G are unique or found in other Apo3 family members.…”

“…A high number of white colonies, illustrating association with many different (Ϫ)DNAs, would be expected for an enzyme with low processivity such as Apo3A. Apo3G has a bimodal off rate from ssDNA that includes short and long binding events (62,63) that can reconcile how a processive enzyme can also cause a high number of clones to be mutated.…”