Mammalian DNA polymerase beta can substitute for DNA polymerase I during DNA replication in Escherichia coli.
Mammalian DNA polymerase beta is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containing its cDNA. Since some catalytic functions of DNA polymerase beta and E. coli DNA polymerase I are similar, we wished to determine if DNA polymerase beta could substitute for DNA polymerase I in bacteria. We found that the expression of mammalian DNA polymerase beta in E. coli restored growth in a DNA polymerase I-defective bacterial mutant. Sucrose density gradient analysis revealed that DNA polymerase beta complements the replication defect in the mutant by increasing the rate of joining of Okazaki fragments. These findings demonstrate that DNA polymerase beta, believed to function in DNA repair in mammalian cells, can also function in DNA replication. Moreover, this complementation system will permit study of the in vivo function of altered species of DNA polymerase beta, an analysis currently precluded by the difficulty in isolating mutants in mammalian cells.
Sweasy
J B
JB
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195.
Loeb
L A
LA
D000818:Animals; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular / Q000379:methods; D004256:DNA Polymerase I / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005816:Genetic Complementation Test; D008322:Mammals; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D004256:DNA Polymerase I