Browsing by Subject "Replication Protein A (RPA)"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Acetylation of Replication Protein A (RPA) Improves its DNA Binding Property
    (Office of the Vice Chancellor for Research, 2016-04-08) Surendran, Sneha; Ononye, Onyeka; Balakrishnan, Lata
    Genome maintenance is critical for cellular survival and growth. Replication Protein A (RPA), a single-strand DNA (ssDNA) binding protein, is vital for various aspects of genome maintenance such as replication, recombination, repair and checkpoint activation. RPA binding to ssDNA protects it from degradation by cellular nucleases, prevents secondary structure formation and from illegitimate recombination. Within the cell, RPA is subject to many post-translational modifications including phosphorylation, SUMOylation and ribosylation. These modifications regulate the activity of RPA with DNA and other binding partners. RPA has been reported to be also modified by acetylation. We found that human RPA (hRPA) can be in vitro acetylated by p300, an acetyl transferase (AT). To study the effect of this modification on its ssDNA binding function, we made use of electro-mobility gel shift assay (EMSA) and bio-layer interferometry (BLI) technology. Using various length oligos, we tested the binding property of unmodified and acetylated RPA. Our results showed that acetylation of RPA increased its binding affinity compared to unmodified RPA. Interestingly, the acetylated form was also able to bind more stably to shorter length oligos compared to the unmodified form. This suggests that the acetylation of RPA improves its ssDNA binding function. This alteration in its enzymatic activity would have significant implications in maintenance of genome fidelity since improved DNA binding function of RPA will protect the genome from both endogenous and exogenous stresses. Additionally, using mass spectrometry analysis we have identified the lysine residues that get modified by the acetyl group both in vitro and in vivo. We are currently studying the factors that trigger this post-translational modification in the cell.
  • Thumbnail Image
    Item
    Biochemical impact of p300-mediated acetylation of replication protein A: Implications for DNA metabolic pathway choice
    (Elsevier, 2025) Ononye, Onyekachi; Surendran, Sneha; Battapadi, Tripthi; VanderVere-Carozza, Pamela; Howald, Olivia K.; Kantartzis-Petrides, Athena; Jordan, Matthew R.; Ainembabazi, Diana; Wold, Marc S.; Turchi, John J.; Balakrishnan, Lata; Biology, School of Science
    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 suppresses illegitimate recombination. In our current study, we identified the acetyltransferase p300 to be capable of acetylating the 70 kDa subunit of RPA in vitro and within cells. The acetylation status of RPA changes throughout the cell cycle, increasing during the S and G2/M phases, and after 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. Enzymatic assays further revealed that upon acetylation RPA governs the switch between the short and long flap pathway for Okazaki fragment processing. Our findings demonstrate that p300-dependent, site-specific acetylation enhances RPA's DNA binding properties, potentially regulating its function during various DNA transactions.