The construct was verified by sequencing (Baseclear, Netherlands). The pFastbac Dual with CSA and DDB1 was transformed into the strain DH10EMBacY (Bergeret al., 2004), kindly provided by Imre Berger (EMBL, France). resolution on beamline ID14-1 at the European Synchrotron Radiation Facility. == 1. Introduction == The human genome is constantly subjected to damaging agents such as ultraviolet irradiation. One of the major pathways for removal of DNA lesions is nucleotide excision repair (NER), which is responsible for the removal of a wide variety of chemically and structurally diverse lesions. A subpathway of NER is transcription coupled NER (TC-NER), which removes lesions blocking transcription. Most proteins are shared between global genome NER (GG-NER) Rabbit Polyclonal to SIN3B and TC-NER, but two proteins are unique to TC-NER: Cockayne syndrome proteins A and B (CSA and CSB). These proteins have been proposed to have the following functions (Fousteriet al., 2006). When RNA polymerase II encounters a DNA lesion during transcription, it becomes stalled and this stabilizes its interaction with CSB. This leads to recruitment of the core NER factors, a chromatin remodeller (HAT p300) as well as CSA in complex with DNA damage-binding protein 1 (DDB1) and E3-ubiquitin-ligase/CSN, which in turn recruits a chromatin remodeller (HMGN1), a scaffolding factor for protein-complex formation (XAB2) and a transcription cleavage factor (TFIIS). The recruitment of the CSADDB1E3-ubiquitin-ligase/CSN complex, which is inactive for E3-ubiquitin-ligase activity upon recruitment, might be to prevent the degradation of the stalled RNA polymerase and/or other TC-NER factors in the early stages of repair, or it might be involved in the degradation of CSB at a later stage of the repair process. The degradation of CSB may be important for recovery of transcription after TC-NER is completed (Groismanet al., 2006). The biological importance of CSA and CSB can be seen since mutations in either protein can cause the recessive human disorder Cockayne syndrome. This disease, named after the London physician Edward Alfred Cockayne (18801956), is characterized by neurologic abnormality, growth retardation, abnormal sensitivity to sunlight and premature aging (Nance & Berry, 1992). 18 different mutations in CSA that can cause Cockayne syndrome (Laugelet al., 2010) have been reported. Cockayne syndrome protein A is found in the cell in complex with DDB1, which is a multifunctional protein that links several different substrate adaptors such as CSA to the Cul4ARoc1 complex, hence constituting the E3-ubiquitin-ligase complex (recently reviewed in Iovineet al., LGD-6972 2011). Several crystal structures of DDB1 have been reported, the first of which was reported by Liet al.(2006). However, to date no structural information is available on CSA. Determination of the structure of Cockayne syndrome protein A will provide insight into the important DNA-repair mechanism TC-NER and into the molecular basis by which the disease-causing mutations in CSA cause Cockayne syndrome. To this end, we report the overproduction, purification and crystallization of CSA in complex with DDB1. == 2. Materials and methods == == 2.1. Cloning and overproduction == The open reading frame (ORF; amino acids 1396) for human CSA (ERCC8; OMIM 609412) was amplified by PCR from a clone in a pFastbac vector kindly made available by Wim Vermeulen (Erasmus Medical Center, The Netherlands) and cloned into the pETM series of vectors (Dmmleret al., 2005) and into pET52b (Invitrogen). Overproduction inEscherichia colistrains BL21 Rosetta, RIL, RP, pLysS and pLysS Star was attempted at 277, 293 and 310 K with 0.11 mMIPTG. Overproduction inE. coliwas concluded to be unsuccessful, after which overproduction of CSA together with its interaction partner DDB1 was attempted in Sf9 insect cells. For this, a vector with the ORF (amino acids 11140) for human DDB1 (OMIM 600045) was kindly made available by Andrea Scrima and Nicolas Thom (Friedrich Miescher Institute, Switzerland). The sequence for an N-terminal 6His tag and a thrombin cleavage site were inserted in front of the ORF LGD-6972 and this sequence was placed behind the p10 promoter in a pFastbac Dual vector (Invitrogen) usingXmaI andAcc65I. The resulting DDB1 molecule had the following extra residues at its N-terminus: MHHHHHHRRLVPRGSGGR. CSA was amplified from the pET52b construct with a C-terminal 10His tag and thrombin cleavage site using the primers 5-TTT CAC GGT CCG GGG ATG CTG GGG TTT TTG TCC GCA LGD-6972 CG-3 and 5-AGT AGT CGA CGT TAA TTA GTG GTG GTG ATG GTG ATG ATG GTG-3 and cloned into the same pFastbac Dual vector as DDB1 behind the PolH promoter using the restriction enzymesRsrII andSalI. The resulting protein lacked the C-terminal glycine and has the following extra residues at its C-terminus: LALVPRGSSAHHHHHHHHHH. The construct was verified by sequencing (Baseclear, Netherlands). The pFastbac Dual with CSA and DDB1 was transformed into the strain DH10EMBacY (Bergeret LGD-6972 al., 2004), kindly provided by Imre Berger (EMBL, France). The recombinant bacmid was isolated and transfected into Sf9 insect cells using Fugene HD (Roche) following the manufacturers instructions. Recombinant baculovirus was produced and insect.