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RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes

Nature volume 412, pages 456–461 (2001)Cite this article

Abstract

Extensive work on the maturation of lagging strands during the replication of simian virus 40 DNA suggests that the initiator RNA primers of Okazaki fragments are removed by the combined action of two nucleases, RNase HI and Fen1, before the Okazaki fragments join1,2,3,4,5. Despite the well established in vitro roles of these two enzymes6, genetic analyses in yeast revealed that null mutants of RNase HI and/or Fen1 are not lethal7,8,9, suggesting that an additional enzymatic activity may be required for the removal of RNA. One such enzyme is the Saccharomyces cerevisiae Dna2 helicase10,11,12/endonuclease12, which is essential for cell viability13,14 and is well suited to removing RNA primers of Okazaki fragments15. In addition, Dna2 interacts genetically and physically with several proteins involved in the elongation or maturation of Okazaki fragments10,16. Here we show that the endonucleases Dna2 and Fen1 act sequentially to facilitate the complete removal of the primer RNA. The sequential action of these enzymes is governed by a single-stranded DNA-binding protein, replication protein-A (RPA). Our results demonstrate that the processing of Okazaki fragments in eukaryotes differs significantly from, and is more complicated than, that occurring in prokaryotes. We propose a novel biochemical mechanism for the maturation of eukaryotic Okazaki fragments.

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Figure 1: RPA has a genetic interaction with Dna2 and stimulates endonuclease activity of Dna2.
Figure 2: RPA and Dna2 form a ternary complex with 5′-tailed DNA substrates.
Figure 3: RPA mediates the sequential and ordered action of Dna2 and Fen1 in the processing of Okazaki fragments.
Figure 4: A model of processing of Okazaki fragments in eukaryotes.

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Acknowledgements

We thank J. Hurwitz, S. MacNeill and A.-K. Bielinsky for critical reading of the manuscript. This work was supported by a grant from the Creative Research Initiatives of the Korean Ministry of Science and Technology given to Y.-S.S.

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  1. National Creative Research Initiative Center for Cell Cycle Control, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chunchun-Dong, Changan-Ku, Suwon, 440-746, Kyunggi-Do, Korea

    Sung-Ho Bae, Kwang-Hee Bae, Jung-Ae Kim & Yeon-Soo Seo

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Correspondence to Yeon-Soo Seo.

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Bae, SH., Bae, KH., Kim, JA. et al. RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes. Nature 412, 456–461 (2001). https://doi.org/10.1038/35086609

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