PURIFICATION AND CHARACTERIZATION OF DNA-POLYMERASE-ZETA FROM MATURE OOCYTES OF THE BONY FISH MISGURNUS-FOSSILIS L (LOACH)

A preparation of DNA polymerase zeta possessing 3'-->5'-exonuclease ac tivity and free from other DNA polymerases was purified from mature oo cytes of the bony fish Misgurnus fossilis L. (loach). The 3'-->5'-exon uclease was cosedimented with DNA polymerase zeta in a 15-30% glycerol gradient with sedimentation coefficient 6S. The preparation contained three major peptides visualized after SDS-PAGE. The amount of one wit h apparent molecular mass of 70 kD was proportional to DNA polymerase and 3'-->5'-exonuclease activities after centrifugation in the glycero l gradient. The 70-kD (pI = 6.3) polypeptide was separated from cosedi mented peptides by two-dimensional electrophoresis. To understand the role of DNA polymerase zeta, properties of the enzyme were studied and compared with reports on other DNA polymerases. DNA polymerase zeta, like DNA polymerase beta, is a low processivity enzyme both in the pre sence and in the absence of PCNA. However, in contrast to DNA polymera se beta, the 3'-->5'-exonuclease activity of DNA polymerase 5 was abno rmally high. 3'-->5'-Exonuclease activity normalized per polymerase ac tivity was 20 times higher in DNA polymerase 5 than in human DNA polym erase epsilon. The exonuclease of DNA polymerase zeta hydrolyzed singl e-stranded poly- and oligonucleotides from their 3'-end. It also remov ed unpaired nucleotides from the 3'-end of the duplex, but hydrolysis was 3 times slower in this case. Further hydrolysis of H-3-labeled nuc leotides paired in duplex was not observed. The distributive type of t he reaction was proved by the kinetics of exonuclease hydrolysis of ol igo-dT(10). All these properties suggest that the DNA polymerase zeta from mature leach oocytes is a repair enzyme with pronounced proofread ing activity.