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The Correcting Role of Autonomous 3" → 5" Exonucleases Contained in Mammalian Multienzyme DNA Polymerase Complexes

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Abstract

A study was made of the correcting role of autonomous 3" → 5" exonucleases (AE) contained in multienzyme DNA polymerase complexes of rat hepatocytes or calf thymocytes. DNA was synthesized on phage φX174 amber3 or M13mp2 primer–templates, and used to transfect Escherichia coli spheroplasts. Frequencies were estimated for direct and reverse mutations resulting from mistakes made in the course of in vitro DNA synthesis. The error rate of the hepatocyte complex was estimated at 3 · 10–6 with equimolar dNTP, and increased tenfold when proteins accounting for 70% of the total 3" → 5" exonuclease activity of the complex were removed. The fidelity of DNA synthesis was completely restored in the presence of exogenous AE (ε subunit of E. coli DNA polymerase III). Nuclear (Pol δn) and cytosolic (Pol δc) forms of DNA polymerase δ were isolated from calf thymocytes. The former was shown to contain an AE (TREX2) absent from the latter. As compared with Pol δc, Pol δn had a 20-fold higher exo/pol ratio and allowed 4–5 times higher fidelity of DNA synthesis. The error rate of DNA polymerase complexes changed when dNTP were used in nonequimolar amounts.

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Authors and Affiliations

  1. Institute of Nuclear Physics, Russian Academy of Sciences, Gatchina, Leningrad Region, 188300, Russia

    I. V. Shevelev, N. V. Belyakova, T. P. Kravetskaya & V. M. Krutyakov

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  1. I. V. Shevelev
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  2. N. V. Belyakova
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  3. T. P. Kravetskaya
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  4. V. M. Krutyakov
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Shevelev, I.V., Belyakova, N.V., Kravetskaya, T.P. et al. The Correcting Role of Autonomous 3" → 5" Exonucleases Contained in Mammalian Multienzyme DNA Polymerase Complexes. Molecular Biology 36, 857–863 (2002). https://doi.org/10.1023/A:1021694212116

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