Biology subjects

Wold, M. S.

Publications and source records attributed to Wold, M. S..

3 recordsLinked to original sources

bioRxiv2024

Biochemical Impact of p300-Mediated Acetylation of Replication Protein A: Implications for DNA Metabolic Pathway Choice

Replication Protein A (RPA), a single-stranded DNA (ssDNA) binding protein, is vital for various aspects of genome maintenance such as replication, recombination, repair and cell cycle checkpoint activation. Binding of RPA to ssDNA protects it from degradation by cellular nucleases, prevents secondary structure formation and illegitimate recombination. In our current study, we identified the acetyltransferase p300 to be capable of acetylating the 70kDa subunit of RPA in vitro and within cells. The acetylation status of RPA was increased specifically during the G1/S phase of the cell cycle and also following exposure to UV-induced damage. Furthermore, we were able to specifically identify RPA directly associated with the replication fork during the S phase and UV damage to be acetylated. Based on these observations, we evaluated the impact of lysine acetylation on the biochemical properties of RPA. Investigation of binding properties of RPA revealed that acetylation of RPA increased its binding affinity to ssDNA compared to unmodified RPA. The improvement in binding efficiency was a function of DNA length with the greatest increases observed on shorter length ssDNA oligomers. Furthermore, the mechanism of acetylated RPAs increased affinity for ssDNA was shown to be a function of a slower rate of dissociation compared to the unmodified form of the RPA. Our findings demonstrate that p300-dependent, site-specific acetylation enhances RPAs DNA binding properties, potentially regulating its function during various DNA transactions.

biochemistry↗

bioRxiv2022

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

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{leftrightarrow}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.

genetics↗

bioRxiv2018

Dynamics and Selective Remodeling of the DNA Binding Domains of RPA

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-strand DNA (ssDNA) intermediates, activating the DNA damage response, and handing off ssDNA to appropriate downstream players. Six DNA binding domains (DBDs) in RPA promote high affinity binding to ssDNA, but also allow RPA displacement by lower affinity proteins. We have made fluorescent versions of RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA, while RPA as a whole remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. This demonstrates how RPA interacting proteins, with lower ssDNA binding affinity, can access the occluded ssDNA and remodel individual DBDs to replace RPA.\n\nOne Sentence SummaryThe choreography of binding and rearrangement of the individual domains of RPA during homologous recombination is revealed.

biochemistry↗