IDENTIFICATION OF A 3'-]5'-EXONUCLEASE THAT REMOVES CYTOSINE-ARABINOSIDE MONOPHOSPHATE FROM 3' TERMINI OF DNA

Cytosine arabinoside monophosphate (araCMP) at the 3' terminus of DNA constitutes a lesion that impedes further synthesis by DNA polymerase alpha (DNA pol alpha). A biochemical assay has been designed to detect 3'-->5'-exonucleases in cell extracts that remove the 3'-araCMP lesio n in an oligonucleotide template primer and permit subsequent extensio n by DNA pol alpha. The major 3'-->5'-exonuclease activity in human my eloblast extracts has been purified, and gel filtration chromatography of the purified enzyme indicates that the exonuclease has an apparent native molecular mass of 52 kDa. Incubation of the enzyme with a 5'-P -32-labeled araCMP template-primer results in exonucleolytic degradati on of the primer exclusively in the 3'-->5' direction, demonstrating t hat the enzyme is a 3'-->5'-exonuclease. The products of the 3'-->5'-e xonuclease reaction are 5'-mononucleotides. The apparent rate of araCM P removal by the exonuclease is approximately the same as the rate of deoxynucleoside monophosphate (dNMP) removal. Furthermore, the apparen t rates of 3'-terminal excision are approximately the same whether the oligomer is hybridized to a complementary oligonucleotide, or not, in dicating that the enzyme has both single- and double-stranded 3'-->5' exonuclease activity. The enzyme does not possess 5'-->3'-exonuclease activity, nor is it associated with DNA polymerase activity. In additi on, the enzyme does not cleave 3'-phosphoryl-terminated DNA, and it do es not cleave RNA. The enzymatic characteristics of the isolated 3'--> 5'-exonuclease indicate that it is distinct from previously identified mammalian deoxyribonucleases.