Mammalian DNA polymerase beta can substitute for DNA polymerase I during DNA replication in Escherichia coli.

J B Sweasy,L A Loeb

doi:10.1016/s0021-9258(18)45956-x

J B Sweasy, L A Loeb

Open Access

https://doi.org/10.1016/s0021-9258(18)45956-x

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Abstract

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.

Highlights

Mammalian DNApolymerase8 is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containingits cDNA
A718polA12" double mutant is unable to grow on semi-enriched orrich media at 42 "C.Significant growth is only detected on these media with the double mutant if it We developed a complementation system in a Pol I-defec- contains thegene encoding pol @T.hese resultsshow that pol tive E. coli mutant
Expression of DNA pol @ and E. coli DNA Pol I, which is encoded by the pol @ in the recA+polA12'"mutant results in the majority of pol4 gene, are postulated to have functional similarities, we nascent DNA being founadt the bottomof the gradient after surmised that transforming therecA718 polAl2'" strain with 4 min, indicating that the fragments are joinemdore rapidly a plasmid that overproduced pol@ might allow this mutant to (Fig. ZC), possibly with a rate surpassing thaotf the wild type grow onrich medium at 42 "C.As showninTable I, the strain (Fig. 2 A )

Summary

Introduction

Mammalian DNApolymerase8 is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containingits cDNA. I n uitro, Pol I is able to catalyze the filling of gaps and to perform nick translation, utilizing its polymerase and 5' to 3' exonuclease activities.

Results

Conclusion