Antagonistic roles of canonical and alternative RPA in tandem CAG repeat diseases

Abstract

Tandem CAG repeat expansion mutations cause >15 neurodegenerative diseases, where ongoing expansions in patients brains are thought to drive disease onset and progression. Repeat length mutations will involve single-stranded DNAs prone to form mutagenic DNA structures. However, the involvement of single-stranded DNA binding proteins (SSBs) in the prevention or formation of repeat instability is poorly understood. Here, we assessed the role of two SSBs, canonical RPA (RPA1-RPA2-RPA3) and the related Alternative-RPA (Alt-RPA, RPA1-RPA4-RPA3), where the primate-specific RPA4 replaces RPA2. RPA is essential for all forms of DNA metabolism, while Alt-RPA has undefined functions. RPA and Alt-RPA are upregulated 2- and 10-fold, respectively, in brains of Huntington disease (HD) and spinocerebellar ataxia type 1 (SCA1) patients. Correct repair of slipped-CAG DNA structures, intermediates of expansion mutations, is enhanced by RPA, but blocked by Alt-RPA. Slipped-DNAs are bound and melted more efficiently by RPA than by Alt-RPA. Removal of excess slipped-DNAs by FAN1 nuclease is enhanced by RPA, but blocked by Alt-RPA. Protein-protein interactomes (BioID) reveal unique and shared partners of RPA and Alt-RPA, including proteins involved in CAG instability and known modifiers of HD and SCA1 disease. RPA overexpression inhibits rampant CAG expansions in SCA1 mouse brains, coinciding with improved neuron morphology and rescued motor phenotypes. Thus, SSBs are involved in repeat length mutations, where Alt-RPA antagonistically blocks RPA from suppressing CAG expansions and hence pathogenesis. The processing of repeat length mutations is one example by which an Alt-RPA-RPA antagonistic interaction can affect outcomes, illuminating questions as to which of the many processes mediated by canonical RPA may also be modulated by Alt-RPA.

Replication Protein A, the Main Eukaryotic Single-Stranded DNA Binding Protein, a Focal Point in Cellular DNA Metabolism

“…These findings suggested that since Alt-RPA has a widely overlapping protein interaction pattern with canonical RPA its expression in cells might be a disease-relevant phenotype [32,33]. This hypothesis was supported by recent findings that Alt-RPA expression supported CAG expansions in neurodegenerative diseases whereas canonical RPA suppressed them suggesting a Yin and Yang relationship between Alt-RPA and canonical RPA in the modulation of CAG repeat instability [34].…”

Human hnRNPA1 reorganizes telomere-bound Replication Protein A

“…Our ability to detect RPA E240K in the hnRNPA1 pull-down, but not in the MP measurements likely reflects an IP of a DNA-mediated complex, as the RPA-hnRNPA1 or RPA E240K -hnRNPA1 complex may protect telomeric ssDNA from micrococcal nuclease digestion. While this work was in revision, BioID experiments confirmed that RPA and hnRNPA1 interact in the cell or are found in a close proximity to one another 37 .…”

“…It is notable that while RPA and hnRNPA1 physically interact 37 , the ternary RPA-DNA-hnRNPA1 complex is specific to telomeric G-quadruplex DNA, as hnRNPA1 was unable to remodel RPA bound to non-structured ssDNA, or to G-quadruplexes formed in the promoters of MYC and BCL-2 genes. These non-telomeric quadruplexes play important regulatory functions in gene expression and can also have pathological roles by interfering with DNA replication and repair.…”

Delineating regional vulnerability in the neurodegenerative disease SCA1 using a conditional mutant ATXN1 mouse

“…Our findings indicate that a relative time course of disease in cerebellum and striatum in f-ATXN1 146Q/2Q mice parallel the MRI findings in SCA1 patients. It is intriguing that there is somatic instability of expanded ATXN1 in the striatum of both SCA1 knockin mice and SCA1 patients 21,22 . We speculate that striatal cells are less sensitive to expanded ATXN1 than are Purkinje cells, requiring the somatic expansion with age for initiation of striatal pathogenesis.…”