Functional consequences of mutations in the conserved SF2 motifs and post-translational phosphorylation of the CSB protein

Abstract

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.

The role of Cockayne Syndrome group B (CSB) protein in base excision repair and aging

“…The SNF-2 like ATPase domain is critical for CSB-catalyzed ATPase activity, but the ATPase activity is differentially affected by mutations in the different domains. Thus, a motif II mutant is more affected than motif V and VI mutants, indicating that motif II is the most important one for the catalytic activity of CSB among the motifs (Christiansen et al, 2003).…”

“…The observation that CSB protein is dephosphorylated at serine and/or threonine in vivo in cells which have been UV-irradiated and that dephosphorylation of CSB stimulates CSB-catalyzed ATPase activity in vitro (Christiansen et al, 2003) suggests that phosphorylation of CSB may regulate its activity in cells with DNA damage. Furthermore, the CSB catalyzed annealing activity is also sensitive to the phosphorylation state of CSB as the strand annealing reaction is stimulated by dephosphorylation of CSB (Muftuoglu et al, 2006).…”

“…The family contains members involved in transcription regulation, chromosome stability, DNA repair and recombination (Eisen et al, 1995). A common feature of SWI/SNF-2 proteins is destabilization of protein-DNA interactions (reviewed in (Christiansen et al, 2003)). As described above, several studies indicate involvement of CSB in transcription regulation, TC-NER and BER of some oxidative lesions.…”

“…Together, the shortening and the topological change argue that CSB, upon ATP binding, wraps around DNA, in a manner similar to a nucleosome. DNA binding and ATPase assays indicate that binding and hydrolysis of ATP result in DNA binding and release (Beerens et al, 2005;Christiansen et al, 2003), although it is not clear whether it is ATP binding or hydrolysis that results in DNA binding. This mechanism could imply that CSB tracks along DNA and alters its topology as a result of the wrapping.…”

Defective Human MutY Phosphorylation Exists in Colorectal Cancer Cell Lines with Wild-type MutY Alleles

“…We treated cell extracts from four defective MSS CRC cell lines, VACO411, VACO 425, VACO429, and VACO489, and two MutY-proficient cell lines, VACO9 M and SW837 (12), with three protein kinases and monitored the effect they had on the cleavage of a 20-bp A⅐8-oxoG oligonucleotide duplex. All three kinases, PKA, PKC, and CKII, are serine/threonine kinases and have been shown to regulate the activities of several DNA repair proteins such as apurinic/apyrimidinic endonuclease 1 (APE1), cockayne syndrome B protein), mutS homolog 2 (MSH2), and replication protein A (RPA) by phosphorylation (17)(18)(19)(20). In vitro phosphorylation of the extracts from all four defective cell lines with these kinases increased the cleavage of the A⅐8-oxoG oligonucleotide considerably, with the most significant increase observed with PKC treatment (Fig.…”