Nucleic acid binding activity of human Cockayne syndrome B protein and identification of Ca(2+) as a novel metal cofactor.
The Cockayne syndrome group B protein (CSB) is a member of the SWI/SNF2 subgroup of Superfamily 2 ATPases/nucleic acid translocases/helicases and is defective in the autosomal recessive segmental progeroid disorder Cockayne syndrome. This study examines the ATP-dependent and the ATP-independent biochemical functions of human CSB. We show that Ca(2+) is a novel metal cofactor of CSB for ATP hydrolysis, mainly through the enhancement of k(cat), and that a variety of biologically relevant model nucleic acid substrates can function to activate CSB ATPase activity with either Mg(2+) or Ca(2+) present. However, CSB lacked detectable ATP-dependent helicase and single- or double-stranded nucleic acid translocase activities in the presence of either divalent metal. CSB was found to support ATP-independent complementary strand annealing of DNA/DNA, DNA/RNA, and RNA/RNA duplexes, with Ca(2+) again promoting optimal activity. CSB formed a stable protein:DNA complex with a 34mer double-stranded DNA in electrophoretic mobility-shift assays, independent of divalent metal or nucleotide (e.g. ATP). Moreover, CSB was able to form a stable complex with a range of nucleic acid substrates, including bubble and "pseudo-triplex" double-stranded DNAs that resemble replication and transcription intermediates, as well as forked duplexes of DNA/DNA, DNA/RNA, and RNA/RNA composition, the latter two of which do not promote CSB ATPase activity. Association of CSB with DNA, independent of ATP binding or hydrolysis, was seemingly sufficient to displace or rearrange a stable pre-bound protein:DNA complex, a property potentially important for its roles in transcription and DNA repair.
Berquist
Brian R
BR
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
Wilson
David M
DM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003057:Cockayne Syndrome / Q000235:genetics*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D009696:Nucleic Acids / Q000378:metabolism*; D000075223:Poly-ADP-Ribose Binding Proteins
D009696:Nucleic Acids; D000075223:Poly-ADP-Ribose Binding Proteins; D000255:Adenosine Triphosphate; D004265:DNA Helicases; C078673:ERCC6 protein, human; D045643:DNA Repair Enzymes; D008274:Magnesium; D002118:Calcium