affinity-purification-6x-his-tagged-protein-using-fast-protein-liquid

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. ...

analyzing-dna-protein-interactions-with-streptavidin-based-biolayer

Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of ...

Indiana University Indianapolis

CTC1-STN1-TEN1 (CST) is a single-stranded DNA binding protein vital for telomere length maintenance with additional genome-wide roles in DNA replication and repair. While CST was previously shown to function in double-strand break repair and promote replication restart, it is currently unclear whether it has specialized roles in other DNA repair pathways. Proper and efficient repair of DNA is critical to protecting genome integrity. Telomeres and other G-rich regions are strongly predisposed to oxidative DNA damage in the form of 8-oxoguanines, which are typically repaired by the base-excision repair (BER) pathway. Moreover, recent studies suggest that CST functions in the repair of oxidative DNA lesions. Therefore, we tested whether CST interacts with and regulates BER protein activity. Here, we show that CST robustly stimulates proteins involved in BER, including OGG1, Pol β, APE1, and LIGI, on both telomeric and non-telomeric DNA substrates. Biochemical reconstitution of the pathway indicates that CST stimulates BER. Finally, knockout of STN1 or CTC1 leads to increased levels of 8-oxoguanine, suggesting defective BER in the absence of CST. Combined, our results define an undiscovered function of CST in BER, where it acts as a stimulatory factor to promote efficient genome-wide oxidative repair.

Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage.

The ATR kinase responds to elevated levels of single-stranded DNA (ssDNA) to activate the G2/M checkpoint, regulate origin utilization, preserve fork stability, and allow DNA repair towards ensuring genome integrity. The intrinsic replication stress in cancer cells makes this pathway an attractive therapeutic target. The ssDNA that drives ATR signaling is sensed by the ssDNA-binding protein replication protein A (RPA), which acts as a platform for ATRIP recruitment and subsequent ATR activation by TopBP1. We have developed chemical RPA inhibitors (RPAi) that block RPA-ssDNA interactions, termed RPA-DBi, and RPA protein-protein interactions, termed RPA-PPIi; both activities are required for ATR activation. Here, we employ a biochemically reconstituted ATR kinase signaling pathway and demonstrate that both RPA-DBi and RPA-PPIi abrogate ATR-dependent phosphorylation of downstream target proteins. We demonstrate that RPA post-translational modifications (PTMs) impact ATR kinase activation but do not alter sensitivity to RPAi. Specifically, phosphorylation of RPA32 and TopBP1 stimulate, while RPA70 acetylation has no effect on ATR phosphorylation of target proteins. Collectively, this work reveals the RPAi mechanism of action to inhibit ATR signaling that can be regulated by RPA PTMs and offers insight into the anti-cancer activity of ATR pathway targeted cancer therapeutics.

The Effect of Replication Protein A Inhibition and Post-Translational Modification on ATR Kinase Signaling.

The ATR kinase responds to elevated levels of single-stranded DNA (ssDNA) to activate the G2/M checkpoint, regulate origin utilization, preserve fork stability, and allow DNA repair to ensure genome integrity. The intrinsic replication stress in cancer cells makes this pathway an attractive therapeutic target. The ssDNA that drives ATR signaling is sensed by the ssDNA-binding protein replication protein A (RPA), which acts as a platform for ATRIP recruitment and subsequent ATR activation by TopBP1. We have developed chemical RPA inhibitors (RPAi) that block RPA-ssDNA interactions (RPA-DBi) and RPA protein-protein interactions (RPA-PPIi); both activities are required for ATR activation. Here, we biochemically reconstitute the ATR kinase signaling pathway and demonstrate that RPA-DBi and RPA-PPIi abrogate ATR-dependent phosphorylation of target proteins with selectivity advantages over active site ATR inhibitors. We demonstrate that RPA post-translational modifications (PTMs) impact ATR kinase activation but do not alter sensitivity to RPAi. Specifically, phosphorylation of RPA32 and TopBP1 stimulate, while RPA70 acetylation does not affect ATR phosphorylation of target proteins. Collectively, this work reveals the RPAi mechanism of action to inhibit ATR signaling that can be regulated by RPA PTMs and offers insight into the anti-cancer activity of ATR pathway-targeted cancer therapeutics.

ArticleTotal : 2	Year
Publication title Cited by 2	2024
Publication title	2025

Article	Year
Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage. Journal of molecular biology\| PubMed ID: 38908783	2024
The Effect of Replication Protein A Inhibition and Post-Translational Modification on ATR Kinase Signaling. Research square\| PubMed ID: 39108493	2024
The effect of replication protein A inhibition and post-translational modification on ATR kinase signaling. Scientific reports\| PubMed ID: 39187637	2024

Lata Balakrishnan

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