DROSOPHILA RIBOSOMAL-PROTEIN PO CONTAINS APURINIC APYRIMIDINIC ENDONUCLEASE ACTIVITY/

Drosophila ribosomal protein PO was overexpressed in Escherichia coli to allow for its purification, biochemical characterization and to gen erate polyclonal antibodies for Western analysis, Biochemical tests we re originally performed to see if overexpressed PO contained DNase act ivity similar to that recently reported for the apurinic/apyrimidinic (AP) lyase activity associated with Drosophila ribosomal protein S3, T he overexpressed ribosomal protein was subsequently found to act on AP DNA, producing scissions that were in this case 5' of a baseless site instead of 3', as has been observed for S3, As a means of confirming that the source of AP endonuclease activity was in fact due to PO, glu tathione S-transferase (GST) fusions containing a Factor Xa cleavage s ite between GST and PO were constructed, overexpressed in an E.coli st rain defective for the major 5'-acting AP endonucleases and the fusion s purified using glutathione-agarose affinity column chromatography, I solated fractions containing purified GST-PO fusion proteins were subs equently found to have authentic AP endonuclease activity, Moreover, g lutathione-agarose was able to deplete AP endonuclease activity from G ST-PO fusion protein preparations, whereas the resin was ineffective i n lowering DNA repair activity for PO that had been liberated from the fusion construct by Factor Xa cleavage, These results suggested that PO was a multifunctional protein with possible roles in DNA repair bey ond its known participation in protein translation. In support of this notion, tests were performed that show that GST-PO, but not GST, was able to rescue an E.coli mutant lacking the major 5'-acting AP endonuc leases from sensitivity to an alkylating agent, We furthermore show th at GST-PO can be located in both the nucleus and ribosomes, Its nuclea r location can be further traced to the nuclear matrix, thus placing P O in a subcellular location where it could act as a DNA repair protein , Other roles beyond DNA repair seem possible, however, since GST-PO a lso exhibited significant nuclease activity for both single- and doubl e-stranded DNA.