DNASEY - A RAT DNASEI-LIKE GENE CODING FOR A CONSTITUTIVELY EXPRESSEDCHROMATIN-BOUND ENDONUCLEASE

A rat gene, designated DNaseY, encoding a 36 kDa endonuclease was iden tified and cloned. Sequence analysis of the cDNA showed it to be the r at homologue of human DNAS1L3. The DNaseY gene product had 42% identit y to DNaseI, including conserved critical active site residues, the es sential disulfide bridge, the calcium binding domain, and a signal pep tide, as well as 2 of the 3 signature boxes. Significantly, DNaseY had 2 nuclear localization signals and was more basic (pI 9.5) than DNase I (pI 4.8). The DNaseY gene contained a number of exons similar to tha t of DNaseI, separated by much larger introns, resulting in a gene of >17 kb compared to <4 kb gene of DNaseI. The 36 kDa DNaseY gene produc t was catalytically inactive but was converted to an active 33 kDa end onuclease following processing of the hydrophobic signal peptide. Anti body generated against peptides representing the predicted amino acid sequence of DNaseY cross-reacted with a 33 kDa nuclear protein which p ossessed endonucleolytic activity. The enzyme was active over a broad pH range (optimum pH 7-8), was Ca2+/Mg2+-dependent, was inhibited by Z n2+ and was capable of both single- and double-stranded DNA cleavage, producing DNA fragments with 3'-OH ends. Furthermore, the DNaseY gene was expressed constitutively in all cells and tissues tested, but it w as not transcriptionally up-regulated in apoptotic cells. All these fe atures were consistent with a role in the early stages of apoptotic DN A fragmentation.