Replication protein A (RPA) hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA

• Published in: PloS one Vol. 6; no. 9; p. e24848
• Main Authors: Lada, Artem G, Waisertreiger, Irina S-R, Grabow, Corinn E, Prakash, Aishwarya, Borgstahl, Gloria E O, Rogozin, Igor B, Pavlov, Youri I
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 15.09.2011; Public Library of Science (PLoS)
• Subjects: Analysis; APOBEC-3G Deaminase; Biology; Blotting, Western; Cancer; Cell Line; Cloning; Cytidine Deaminase - genetics; Cytidine Deaminase - metabolism; Cytosine; Cytosine deaminase; Deamination; Deoxyribonucleic acid; Diversification; DNA; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; Editing; Electrophoretic Mobility Shift Assay; Enzymes; Gene loci; Genetic aspects; Genetic research; Genomes; Genomics; Humans; Immunoglobulins; Immunohistochemistry; Kinases; Laboratories; Microscopy, Fluorescence; Mutagenesis; Mutation; Oligonucleotides; Phosphorylation; Physiological aspects; Protein A; Protein binding; Proteins; Pyrimidines; Replication; Replication protein A; Replication Protein A - genetics; Replication Protein A - metabolism; Single-stranded DNA; Steric hindrance; Yeast; United States > US; Nebraska
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0024848

Editing 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. We 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. Our 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.