Functional consequences of mutations in the conserved SF2 motifs and post-translational phosphorylation of the CSB protein.
The rare inherited human genetic disorder Cockayne syndrome (CS) is characterized by developmental abnormalities, UV sensitivity and premature aging. The cellular and molecular phenotypes of CS include increased sensitivity to UV-induced and oxidative DNA lesions. Two genes are involved: CSA and CSB. The CS group B (CSB) protein has roles in transcription, transcription-coupled repair, and base excision repair. It is a DNA stimulated ATPase and remodels chromatin in vitro. Here, we have analyzed wild-type (wt) and motif II, V and VI mutant CSB proteins. We find that the mutant proteins display different degrees of ATPase activity deficiency, and in contrast to the in vivo complementation studies, the motif II mutant is more defective than motif V and VI CSB mutants. Furthermore, CSB wt ATPase activity was studied with different biologically important DNA cofactors: DNA with different secondary structures and damaged DNA. The results indicate that the state of DNA secondary structure affects the level of CSB ATPase activity. We find that the CSB protein is phosphorylated in untreated cells and that UV irradiation leads to its dephosphorylation. Importantly, dephosphorylation of the protein in vitro results in increased ATPase activity of the protein, suggesting that the activity of the CSB protein is subject to phosphorylation control in vivo. These observations may have significant implications for the function of CSB in vivo.
Christiansen
Mette
M
Danish Center for Molecular Gerontology, University of Aarhus, DK-8000 Aarhus C, Denmark.
Stevnsner
Tinna
T
Modin
Charlotte
C
Martensen
Pia M
PM
Brosh
Robert M
RM
Bohr
Vilhelm A
VA
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D001483:Base Sequence; D002478:Cells, Cultured; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D004260:DNA Repair; D045643:DNA Repair Enzymes; D006801:Humans; D009154:Mutation; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D000075223:Poly-ADP-Ribose Binding Proteins; D011499:Protein Processing, Post-Translational
D000075223:Poly-ADP-Ribose Binding Proteins; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C078673:ERCC6 protein, human; D045643:DNA Repair Enzymes