Abstract
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 lesion in an oligonucleotide template-primer and permit subsequent extension by DNA pol alpha. The major 3'-->5'-exonuclease activity in human myeloblast 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'-32P-labeled araCMP template-primer results in exonucleolytic degradation of the primer exclusively in the 3'-->5' direction, demonstrating that the enzyme is a 3'-->5'-exonuclease. The products of the 3'-->5'-exonuclease reaction are 5'-mononucleotides. The apparent rate of araCMP removal by the exonuclease is approximately the same as the rate of deoxynucleoside monophosphate (dNMP) removal. Furthermore, the apparent rates of 3'-terminal excision are approximately the same whether the oligomer is hybridized to a complementary oligonucleotide, or not, indicating 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 addition, the enzyme does not cleave 3'-phosphoryl-terminated DNA, and it does not cleave RNA. The enzymatic characteristics of the isolated 3'-->5'-exonuclease indicate that it is distinct from previously identified mammalian deoxyribonucleases.
Highlights
From the Department of Biochemistry and Comprehensive Cancer Center of Wake Forest University Medical Centeer, Winston-Salem, North Carolina27157
3’ terminus of DNA constitutes a lesion that impedes replication or repair could potentially lead to thinecorporation further synthesis by DNA polymerase (Y (DNA pol cy).A of arabinoside nucleotides into DNA
Theenzymedoes DNA polymerases that contain 3’-5‘ proofreading exonuclenot possess 5’+3’-exonucleaseactivity, nor is it associ- ases, the 3“terminal araNMP cabne removed by the exonucleated with DNA polymerase activity
Summary
These data araCMP RepairAssay-The 25-mer-araC primer was hybridized t o indicate that the 3’-5‘-exonuclease has been purified approximately the 35-mer template to produce a DNA duplex containing a dGMP: 10,000-fold from the myeloblast cell extract with a specific activity of araCMP (GaraC)at the 3’ primer terminus (see Fig.1).Reactions (10 74,000 unitdmg of protein (Fraction VII). Reactions were as described An aruCMP Repair Activity I s Detected in Leukemia Cell for the araCMP repair assay, except the 25-mer-araC primer was labeled with 32Pat the 5’ terminus and DNA pol a and dNTPs were
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