Replication Protein A (RPA) Hampers the Processive Action of APOBEC3G Cytosine Deaminase on Single-Stranded DNA

Abstract

BackgroundEditing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G), restricts retroviruses, and Activation Induced Deaminase (AID) generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G.Principal Findings/MethodologyWe found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the “hit and run” single base substitution events observed in yeast.SignificanceOur data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

“…For AID, a physical interaction with RPA occurred and promoted AID-catalyzed deaminations in immunoglobulin variable genes and switch regions [366,367]. However, A3G was characterized to have decreased specific activity and processivity in the presence of RPA [368]. The study with A3G agrees with our data (Figure 10.13C and Table 10.2) and we provide a mechanism for why A3G activity decreases in the presence of RPA.…”

Glucocorticoid Receptor Transcriptional Activation via the BRG1-Dependent Recruitment of TOP2β and Ku70/86

“…These results, however, do not preclude additional functions for the BRG1/TOP2A/B complexes. Indeed, recent experiments demonstrate that in mouse embryonic stem cells and in mouse embryonic fibroblasts, BRG1 and TOP2A function to resolve anaphase bridges, and in cancer cells mutations that impair BRG1 contribute to genomic instability and cancer etiology (76). The elegant demonstration of TOP2A/BRG1 activities in decatenation reveals a clear role for regions of BRG1 where mutations have been shown to be important for tumorigenesis (77).…”

APOBEC3A and APOBEC3B Preferentially Deaminate the Lagging Strand Template during DNA Replication

“…Surprisingly, we observed within rfa1-t33 ung1 Δ strains a significant increase in the number of Can R mutants containing multiple APOBEC-induced mutations (p=0.01 by Fisher's Exact Test) ( Table 1 and Table S1 ). Some Can R isolates contained up to 6 strand-coordinated mutations which were likely induced in a single event, suggesting that loss of full RPA functionality may facilitate the processivity of APOBEC enzymes (Lada et al, 2011, Pham et al, 2007). We tested an additional null strain to determine whether the increased APOBEC-induced mutagenesis observed in the rfa1-t33 strains is generalizable to any deficiency in replication fork stability.…”