Abstract
Cockayne syndrome (CS) is an inherited disorder that involves photosensitivity, developmental defects, progressive degeneration and characteristics of premature aging. Evidence indicates primarily nuclear roles for the major CS proteins, CSA and CSB, specifically in DNA repair and RNA transcription. We reveal herein a complex regulation of CSB targeting that involves three major consensus signals: NLS1 (aa467-481), which directs nuclear and nucleolar localization in cooperation with NoLS1 (aa302-341), and NLS2 (aa1038-1055), which seemingly optimizes nuclear enrichment. CSB localization to the nucleolus was also found to be important for full UVC resistance. CSA, which does not contain any obvious targeting sequences, was adversely affected (i.e. presumably destabilized) by any form of truncation. No inter-coordination between the subnuclear localization of CSA and CSB was observed, implying that this aspect does not underlie the clinical features of CS. The E3 ubiquitin ligase binding partner of CSA, DDB1, played an important role in CSA stability (as well as DDB2), and facilitated CSA association with chromatin following UV irradiation; yet did not affect CSB chromatin binding. We also observed that initial recruitment of CSB to DNA interstrand crosslinks is similar in the nucleoplasm and nucleolus, although final accumulation is greater in the former. Whereas assembly of CSB at sites of DNA damage in the nucleolus was not affected by RNA polymerase I inhibition, stable retention at these sites of presumed repair was abrogated. Our studies reveal a multi-faceted regulation of the intranuclear dynamics of CSA and CSB that plays a role in mediating their cellular functions.
Highlights
Based on identified protein interactions and certain cellular phenotypes, namely the profound sensitivity to ultraviolet C (UVC) radiation of CSA and CSB mutant cells, the Cockayne syndrome (CS) proteins have been proposed to function at the interface of transcription and DNA repair, in facilitating the removal of transcription-blocking lesions, such as ultraviolet (UV) photoproducts, in a process termed transcription-coupled nucleotide excision repair (TC-NER)[9]
Prior work reported that both CSA and CSB localize to the nucleus, with the former predominantly in the nucleoplasm and the latter possibly enriched in the nucleolus[23,24,25,26,27]
These findings are consistent with the CS proteins operating primarily in DNA repair and RNA transcription, perhaps more so for rDNA21
Summary
Based on identified protein interactions and certain cellular phenotypes, namely the profound sensitivity to ultraviolet C (UVC) radiation of CSA and CSB mutant cells, the CS proteins have been proposed to function at the interface of transcription and DNA repair, in facilitating the removal of transcription-blocking lesions, such as ultraviolet (UV) photoproducts, in a process termed transcription-coupled nucleotide excision repair (TC-NER)[9]. The stalled transcriptional events and the associated persistent DNA intermediates are thought to lead to hyperactivation of poly(ADP-ribose) polymerase 1 (PARP1), consequent nicotinamide adenine dinucleotide (NAD+) depletion, and mitochondrial dysfunction, all of which participate in the etiology of CS22. Consistent with their apparent biological roles, cellular localization studies have revealed that CSA exists primarily in the nucleus, with some presence in the nucleolus[23,24,25,26,27]. The aim of the current study was to identify elements of CSA and CSB that regulate the intracellular distribution of the proteins, as well as to determine whether CSB might respond uniquely to DNA damage within the nucleoplasm versus nucleolus
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