Biology subjects

Balakrishnan, L.

Publications and source records attributed to Balakrishnan, L..

4 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

Msh2-Msh3 interferes with DNA metabolism in vivo

Mismatch repair (MMR) is a highly conserved DNA repair pathway that safeguards the genome from errors in DNA replication. In Saccharomyces cerevisiae, two MutS homolog (Msh) complexes, Msh2-Msh3 or Msh2-Msh6, initiate MMR. Msh2-Msh3, the focus of this study, recognizes and directs repair of insertion/deletion loops (IDLs) up to ~17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR), and the DNA damage response. Msh2-Msh3 also promotes genome instability through trinucleotide repeat (TNR) expansions. This non-canonical activity is likely an unfortunate consequence of Msh2-Msh3s intrinsic ability to bind a wide range of DNA structures, including those formed with single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5 ssDNA flap structures interfered with the in vitro binding and cleavage activities of the flap endonuclease Rad27 (Fen1 in mammals), which promotes 5 ssDNA flap processing during Okazaki fragment maturation (OFM) and long-patch base excision repair (LP-BER). Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and LP-BER in vivo, consistent with the hypothesis that protein abundance and Msh3 ATPase activities are key drivers of Msh2-Msh3-mediated genomic instability.

genetics↗

bioRxiv2020

Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

ABSTRACTIn S. cerevisiae, the Pif1 helicase functions to impact both nuclear and mitochondrial DNA replication and repair processes. Pif1 is a 5’-3’ helicase, which preferentially unwinds RNA-DNA hybrids and resolves G-quadruplex structures. Further, regulation of Pif1 by phosphorylation negatively impacts its interaction with telomerase during double strand break repair. Here, we report that in addition to phosphorylation, Pif1 is also modified by lysine acetylation, which influences both its cellular and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 (Esa1) and deacetylase Rpd3 are primarily responsible for dynamically acetylating nuclear Pif1. Mass spectrometry analysis revealed that Pif1 was modified throughout the protein’s sequence on the N-terminus (K118, K129), helicase domain (K525, K639, K725), and C-terminus (K800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N-terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase activity, while maintaining its substrate preferences. Additionally, both the ATPase and DNA binding activities of Pif1 were stimulated upon acetylation. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose an acetylation-based model for the regulation of Pif1 activities, addressing how this post translational modification can influence its role as a key player in a multitude of DNA transactions vital to the maintenance of genome integrity.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2018

Directed nucleosome sliding during the formation of the SV40 virus particle exposes DNA sequences required for early transcription

Simian Virus 40 (SV40) exists as chromatin throughout its life cycle, and undergoes typical epigenetic regulation mediated by changes in nucleosome location and associated histone modifications. In order to investigate the role of epigenetic regulation during the encapsidation of late stage minichromosomes into virions, we have mapped the location of nucleosomes containing acetylated or methylated lysines in the histone tails of H3 and H4 present in the chromatin from 48-hour post-infection minichromosomes and disrupted virions. In minichromosomes obtained late in infection, nucleosomes were found carrying various histone modifications primarily in the regulatory region with a major nucleosome located within the enhancer and other nucleosomes at the early and late transcriptional start sites. The nucleosome found in the enhancer would be expected to repress early transcription by blocking access to part of the SP1 binding sites and the left side of the enhancer in late stage minichromosomes while also allowing late transcription. In chromatin from virions, the principal nucleosome located in the enhancer was shifted ~ 70 bases in the late direction from what was found in minichromosomes and the level of modified histones was increased throughout the genome. The shifting of the enhancer-associated nucleosome to the late side would effectively serve as a switch to relieve the repression of early transcription found in late minichromosomes while likely also repressing late transcription by blocking access to necessary regulatory sequences. This epigenetic switch appeared to occur during the final stage of virion formation.

molecular biology↗