USP7-mediated deubiquitination differentially regulates CSB but not UVSSA upon UV radiation-induced DNA damage

ABSTRACT

Cockayne syndrome group B (CSB) protein participates in transcription-coupled nucleotide excision repair. The stability of CSB is known to be regulated by ubiquitin-specific protease 7 (USP7). Yet, whether USP7 acts as a deubiquitinating enzyme for CSB is not clear. Here, we demonstrate that USP7 deubiquitinates CSB to maintain its levels after ultraviolet (UV)-induced DNA damage. While both CSB and UV-stimulated scaffold protein A (UVSSA) exhibit a biphasic decrease and recovery upon UV irradiation, only CSB recovery depends on USP7, which physically interacts with and deubiquitinates CSB. Meanwhile, CSB overexpression stabilizes UVSSA, but decrease UVSSA’s presence in nuclease-releasable/soluble chromatin, and increase the presence of ubiquitinated UVSSA in insoluble chromatin alongside CSB-ubiquitin conjugates. Remarkably, CSB overexpression also decreases CSB association with USP7 and UVSSA in soluble chromatin. UVSSA exists in several ubiquitinated forms, of which mono-ubiquitinated form and other ubiquitinated UVSSA forms are detectable upon 6xHistidine tag-based purification. The ubiquitinated UVSSA forms, however, are not cleavable by USP7 in vitro. Furthermore, USP7 disruption does not affect RNA synthesis but decreases the recovery of RNA synthesis following UV exposure. These results reveal a role of USP7 as a CSB deubiquitinating enzyme for fine-tuning the process of TC-NER in human cells.

KEYWORDS:

• Ubiquitin-specific protease 7 (USP7)
• nucleotide excision repair (NER)
• transcription-coupled nucleotide excision repair (TC-NER)
• Cockayne syndrome group B (CSB)
• ultraviolet radiation (UVR)-stimulated scaffold protein A (UVSSA)
• valosin-containing protein (VCP)/p97

Acknowledgments

Authors thank Dr. Bert Vogelstein for providing HCT116, HCT116-p53-/- and HCT116-USP7-/- cell lines. Authors are also grateful to Drs. Yanhui Xu at Department of Biochemistry, Fudan University Medical School and Dr. Yang Shi at Department of Cell Biology, Harvard Medical School for providing expression constructs of USP7. This work was supported by Public Health service Grants (ES2388, ES12991) from the National Institute of Health.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary Material

Supplementary data for this article can be accessed here.

Additional information

Funding

This work was supported by the National Institute of Environmental Health Sciences [ES012991].