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Purification and substrate specificity of polydeoxyribonucleotide kinases isolated from calf thymus and rat liver
Author(s) -
KarimiBusheri Feridoun,
Weinfeld Michael
Publication year - 1997
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/(sici)1097-4644(199702)64:2<258::aid-jcb9>3.0.co;2-w
Subject(s) - kinase , substrate (aquarium) , substrate specificity , chemistry , biochemistry , microbiology and biotechnology , biology , enzyme , ecology
Damage to DNA can result in strand breaks with 5′‐hydroxyl and 3′‐phosphate termini. Before DNA polymerases and ligases can rejoin the broken strands, such termini have to be restored to 5′‐phosphate and 3′‐hydroxyl groups. Polydeoxynucleotide kinase is an enzyme that may fulfil this function. We have purified the kinases from calf thymus and rat liver to near homogeneity. Based on SDS‐polyacrylamide gel electrophoresis and activity gels, the enzymes from both sources are ∼60‐kDa polypeptides. Both enzymes have an acidic pH optimum (5.5–6.0) for kinase activity, and similar pl values (8.5–8.6), and a specificity for DNA. The calf thymus kinase possesses a 3′‐phosphatase activity, as has previously been shown for the rat liver enzyme. The minimum size of oligonucleotide that can be labelled is 7–8 nucleotides in length, but the optimal size appears to be >18 nucleotides. Comparison of phosphorylation of oligo(dA) 24 and oligo(dT) 24 with oligonucleotides containing a varied nucleotide sequence indicated that the homopolymers are poorer substrates. Unlike the bacteriophage T4 polynucleotide kinase, the mammalian kinases exhibit no preference for 5′‐overhanging termini when acting at DNA termini produced by restriction enzymes. With double‐stranded oligonucleotide complexes designed to model single‐strand gaps and nicks, the mammalian kinases preferentially phosphorylate the 5′‐terminus associated with the gap or nick, in keeping with the idea that the kinases are involved in the repair of DNA single‐strand breaks. J. Cell. Biochem. 64:258–272. © 1997 Wiley‐Liss, Inc.

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