phrA, the major photoreactivating factor in the cyanobacterium Synechocystis sp strain PCC 6803 codes for a cyclobutane-pyrimidine-dimer-specific DNAphotolyase

A new broad-host-range plasmid, pSL1211, was constructed for the over-expre ssion of genes in Synechocystis sp. strain PCC 6803. The plasmid was derive d from RSF1010 and an Escherichia coli over-expression plasmid, pTrcHisC. O ver-expressed protein is made with a removable N-terminal histidine tag. Th e plasmid was used to over-express the phrA gene and purify the gene produc t from Synechocystis sp. strain PCC 6803. PhrA is the major ultraviolet-lig ht-resistant factor in the cyanobacterium. The purified PhrA protein exhibi ted an optical absorption spectrum similar to that of the cyclobutane pyrim idine dimer (CPD) DNA photolyase from Synechococcus sp. strain PCC 6301 (An acystis nidulans). Mass spectrometry analysis of PhrA indicated that the pr otein contains 8-hydroxy-5-deazariboflavin and flavin adenine dinucleotide (FADH(2)) as cofactors. PhrA repairs only cyclobutane pyrimidine dimer but not pyrimidine (6-4) pyrimidinone photoproducts. On the basis of these resu lts, the PhrA protein is classified as a class I, HDF-type, CPD DNA photoly ase.