Cockayne Syndrome (CS) is an autosomal recessive disorder, characterized by growth failure, neurological abnormalities, premature aging symptoms, and cutaneous photosensitivity, but no increased cancer incidence. CS is divided into two major complementation groups: CSA (mutation in CKN1) and CSB (mutation in ERCC6). Of the patients suffering from CS, 80% have mutations in the CSB gene. Our past work has helped define the biochemical properties of CSB, revealing that the protein interacts with a diverse range of nucleic acid substrates and likely has important ATP-dependent and ATP-independent functions in DNA and/or RNA metabolism. Recently, we have focused our research on delineating interacting partners and localization of the CS proteins. All our findings point to a complex regulation of CSB targeting that involves three main consensus signals which have subsequently been found to be important in full UVC resistance. Enrichment of CSB in the nucleolus has been shown to depend upon NLS1 (aa467-481), in concert with NoLS1 (aa302-341), and NLS2 (aa1038-1055). CSA, however, has so far not been shown to contain any apparent targeting sequences, which can be destabilized by any form of truncation. Also, we did not observe any coordination between CSA and CSB when it comes to subnuclear localization. Thus, this lack of coordination indicates that this feature of the proteins most likely does not relate to the clinical features observed in CS. DDB1, the E3 ubiquitin ligase binding partner of CSA, was found to have an important role in CSA stability, as was DDB2. DDB1 was found to assist with the association of CSA to chromatin post UV irradiation, but it was not found to affect the binding of CSB to chromatin. CSB was found to recruit to interstrand crosslinks within the nucleoplasm and nucleolus in a similar manner, although CSBs final accumulation was found to be greater in the nucleoplasm. Inhibition of RNA polymerase I did not affect the gathering of CSB at areas of damage in the nucleolus, but stabilized retention at these sites was abolished. In summary, we found that CSAs and CSBs cellular functions are controlled by regulation of their intranuclear dynamics. Mutations in CSA, CSB, XPB, XPD, XPG, XPF-ERCC1 give rise to CS. To identify molecular pathways that may shed information on understanding the disease etiology, we conducted yeast two-hybrid (Y2H) screens using full-length or C-terminal (Pro1010-Cys1493) CSB as bait. CSB is a DNA-dependent ATPase and chromatin remodeler, with demonstrated roles in RNA Polymerase II-dependent transcription-coupled nucleotide excision repair. A total of 12 interactors were identified, but only LEO1 (Phe381-Ser568 region) appeared as a CSB binding partner in both screens. LEO1 is a member of the polymerase associated factor 1 complex (PAF1C) with roles in transcription elongation and chromatin modification. We believe that the LEO1-CSB interaction represents a novel link that coordinates RNA transcription with DNA repair. Ongoing work includes investigating further CSB:LEO1 interaction and exploring the recruitment of LEO1-CSB to localized sites of DNA damage following laser irradiation and confocal microscopy. Efforts to characterize this novel interaction for its biological function will shed important insight into the molecular processes that regulate the coordination between transcription and DNA repair and may have implications for age-related disease treatment.
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| Abbotts, Rachel; Wilson 3rd, David M (2017) Coordination of DNA single strand break repair. Free Radic Biol Med 107:228-244 |
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| Iyama, Teruaki; Wilson 3rd, David M (2016) Elements That Regulate the DNA Damage Response of Proteins Defective in Cockayne Syndrome. J Mol Biol 428:62-78 |
| Scheibye-Knudsen, Morten; Tseng, Anne; Borch Jensen, Martin et al. (2016) Cockayne syndrome group A and B proteins converge on transcription-linked resolution of non-B DNA. Proc Natl Acad Sci U S A 113:12502-12507 |
| Scheibye-Knudsen, Morten; Fang, Evandro F; Croteau, Deborah L et al. (2015) Protecting the mitochondrial powerhouse. Trends Cell Biol 25:158-70 |
| Ghosh, Somnath; Canugovi, Chandrika; Yoon, Jeong Seon et al. (2015) Partial loss of the DNA repair scaffolding protein, Xrcc1, results in increased brain damage and reduced recovery from ischemic stroke in mice. Neurobiol Aging 36:2319-2330 |
| Sykora, Peter; Misiak, Magdalena; Wang, Yue et al. (2015) DNA polymerase ? deficiency leads to neurodegeneration and exacerbates Alzheimer disease phenotypes. Nucleic Acids Res 43:943-59 |
| Iyama, Teruaki; Lee, Sook Y; Berquist, Brian R et al. (2015) CSB interacts with SNM1A and promotes DNA interstrand crosslink processing. Nucleic Acids Res 43:247-58 |
| Li, Mengxia; Völker, Jens; Breslauer, Kenneth J et al. (2014) APE1 incision activity at abasic sites in tandem repeat sequences. J Mol Biol 426:2183-98 |
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