Biomedical Sciences ETDs

Publication Date

Spring 5-1-2025

Abstract

The ATP-dependent chromatin remodeler Cockayne syndrome protein B (CSB) is essential for maintaining genome integrity. Mutations in CSB are the primary cause of Cockayne syndrome (CS), a premature aging disorder with severe sun sensitivity and neurodevelopmental symptoms. However, the precise mechanisms underlying the disease etiology remain poorly understood. Given the critical roles of oxidative stress in aging and neurodegenerative disorders, this research sought to determine the mechanisms of CSB’s functions in oxidative DNA damage repair.

In the first aim, we investigated how CSB participates in PARP1/2-mediated oxidative DNA repair. We showed that PARP1 and PARP2 recruit CSB to oxidized chromatin and that CSB, in turn, recruits XRCC1 and histone PARylation factor 1 (HPF1). We also demonstrated that CSB participates in PARP1/2-mediated single-strand break repair pathway and that CSB-mediated SSBR occurs primarily at actively transcribed chromatin regions. Importantly, we also found that PARP2, unlike PARP1, primarily functions in actively transcribed regions, while PARP1 does not show any preference. Therefore, this study proposes a novel oxidative DNA repair pathway called transcription-associated single-strand break repair (TA-SSBR), mediated by CSB.

In the second aim, the mechanism by which CSB dissociates poly(ADP-ribose) polymerase-1/2 (PARP1/2) during transcription-associated single-strand break repair (TA-SSBR) was investigated. In Chapter 2, we demonstrated that CSB participates in PARP1/2-dependent single-strand break repair in actively transcribed chromatin regions (TA-SSBR). However, successful repair requires the removal of PARP1/2 from the DNA lesion. Alkaline comet assay data demonstrated that cells with an engineered CSB deletion mutation [N1 region; aaD(245-365)], lacking chromatin remodeling and PAR-binding abilities but retaining ATPase activity, exhibited increased DNA repair defects and heightened sensitivity to PARP inhibitor KU-0058948 compared to CSBnull and ATPase-defective CSBK538A cells. The repair defect in CSBDN1 cells was rescued by transcription inhibition and reduced PARP1 and PARP2 expression, suggesting the repair defect is associated with TA-SSBR. Study also found that the repair defect in CSBDN1 cells is attributed to its PAR-binding deficiency. In summary, this study suggests that CSB’s ATP-dependent chromatin remodeling activity is triggered by binding to PAR. Without PAR-binding, CSB fails to dissociate PARP1/2, leading to the accumulation of a repair intermediate on the active chromatin, causing TA-SSBR to stall.

Keywords

Cockayne syndrome protein B (CSB), ATP-dependent chromatin remodeler, PARP1, PARP2, DNA repair, Transcription-associated single-strand break repair

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Alan E Tomkinson (Chair)

Second Committee Member

Hua-Ying Fan (Mentor)

Third Committee Member

Mary Ann Osley

Fourth Committee Member

Peng Mao

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