Abstract
Oxidative stress contributes to numerous diseases, including cancer. CSB is an ATP-dependent chromatin remodeler critical for oxidative stress relief. PARP1 is the major sensor for DNA breaks and fundamental for efficient single-strand break repair. DNA breaks activate PARP1, leading to the synthesis of poly(ADP-ribose) (PAR) on itself and neighboring proteins, which is crucial for the recruitment of DNA repair machinery. CSB and PARP1 interact; however, how CSB mechanistically participates in oxidative DNA damage repair mediated by PARP1 remains unclear. Using chromatin immunoprecipitation followed by quantitative PCR, we found that CSB and PARP1 facilitate each other’s chromatin association during the onset of oxidative stress, and that CSB facilitates PARP1 removal when the level of chromatin-bound CSB increases. Furthermore, by monitoring chromatin PAR levels using Western blot analysis, we found that CSB sustains the DNA damage signal initiated by PARP1, and may prevent PARP1 overactivation by facilitating DNA repair. By assaying cell viability in response to oxidative stress, we further demonstrate that PARP1 regulation by CSB is a major CSB function in oxidatively-stressed cells. Together, our study uncovers a dynamic interplay between CSB and PARP1 that is critical for oxidative stress relief.
Highlights
The average human cell is estimated to receive about 20,000 DNA-damaging events daily through reactive oxygen species (ROS) generated from normal metabolic processes [1,2].If not repaired efficiently, DNA damage will lead to genome instability, which can result in cell death or disease, such as cancer
We previously found that poly(ADP-ribose) polymerase 1 (PARP1) positively regulates the recruitment of Cockayne syndrome group B protein (CSB) to these sites, which is to a large degree independent of PARP1 enzymatic activity [22]
To study further how critical PARP1 is in regulating menadione-induced CSB recruitment to these loci, we performed anti-CSB Chromatin immunoprecipitation (ChIP)-qPCR in the near-diploid retinal pigment epithelial (RPE) cell line (PARP1+/+ ) and the isogenic RPE cell line in which both PARP1 alleles were deleted by CRISPR (PARP1−/− ) [32]
Summary
The average human cell is estimated to receive about 20,000 DNA-damaging events daily through reactive oxygen species (ROS) generated from normal metabolic processes [1,2].If not repaired efficiently, DNA damage will lead to genome instability, which can result in cell death or disease, such as cancer. The average human cell is estimated to receive about 20,000 DNA-damaging events daily through reactive oxygen species (ROS) generated from normal metabolic processes [1,2]. The hydroxyl radical is the major cause of ROSinduced DNA damage; it attacks the sugar of the phosphodiester backbone as well as DNA bases. These two types of DNA lesions are repaired by single-strand break repair (SSBR). Initiation of SSBR is achieved by the rapid localization of PARP1 to SSBs. BER starts with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease that generates
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