BIOCHEMICAL AND BIOLOGICAL CHARACTERIZATION OF WILD-TYPE AND ATPASE-DEFICIENT COCKAYNE-SYNDROME B REPAIR PROTEIN

Cockayne syndrome (CS) is a nucleotide excision repair disorder charac terized by sun (UV) sensitivity and severe developmental problems. Two genes have been shown to be involved: CSA and CSB. Both proteins play an essential role in preferential repair of transcription-blocking le sions from active genes. In this study we report the purification and characterization of baculovirus-produced HA-His(6)-tagged CSB protein (dtCSB), using a highly efficient three-step purification protocol. Mi croinjection of dtCSB protein in CS-B fibroblasts shows that it is bio logically functional in vivo. dtCSB exhibits DNA-dependent ATPase acti vity, stimulated by naked as well as nucleosomal DNA. Using structural ly defined DNA oligonucleotides, we show that double-stranded DNA and double-stranded DNA with partial single-stranded character but not tru e single-stranded DNA act as efficient cofactors for CSB ATPase activi ty. Using a variety of substrates, no overt DNA unwinding by dtCSB cou ld be detected, as found with other SNF2/SWI2 family proteins. By site -directed mutagenesis the invariant lysine residue in the NTP-binding motif of CSB was substituted with a physicochemically related arginine , As expected, this mutation abolished ATPase activity. Surprisingly, the mutant protein was nevertheless able to partially rescue the defec t in recovery of RNA synthesis after UV upon microinjection in CS-B fi broblasts, These results indicate that integrity of the conserved nucl eotide-binding domain is important for the in vivo function of CSB but that also other properties independent from ATP hydrolysis may contri bute to CSB biological functions.