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Related Experiment Videos

Junction ribonuclease: an activity in Okazaki fragment processing

R S Murante1, L A Henricksen, R A Bambara

  • 1Department of Biochemistry and Biophysics, and Cancer Center, Box 712, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.

Proceedings of the National Academy of Sciences of the United States of America
|April 16, 1998
PubMed
Summary

RNase HI enzyme recognizes and cleaves RNA-DNA junctions in Okazaki fragments, even with mismatches. This junction RNase activity, distinct from heteroduplex recognition, precedes flap endonuclease 1 (FEN1) removal of the RNA primer.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Okazaki fragment maturation is crucial for DNA replication fidelity.
  • Initiator RNA removal involves RNase HI and flap endonuclease 1 (FEN1).
  • RNase HI's substrate specificity in Okazaki fragment processing remains debated.

Purpose of the Study:

  • To elucidate the substrate recognition mechanism of RNase HI during Okazaki fragment processing.
  • To characterize the 'junction RNase' activity observed in RNase HI preparations.
  • To clarify the sequential roles of RNase HI and FEN1 in initiator RNA removal.

Main Methods:

  • Purification of calf RNase HI to high homogeneity.
  • In vitro cleavage assays using various Okazaki fragment substrates, including those with mismatches and unannealed structures.

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  • Analysis of enzyme activity during purification to assess co-migration of RNase HI and junction RNase activities.
  • Assessment of FEN1 activity on processed substrates.

    • Main Results:

    • Highly purified calf RNase HI exhibits precise cleavage at the RNA-DNA junction of Okazaki fragments, irrespective of heteroduplex integrity.
    • This junction cleavage occurs even on fully unannealed Okazaki fragments, indicating recognition of the RNA-DNA transition.
    • The junction RNase activity co-migrates with RNase HI activity, suggesting a single enzyme possesses both functions.
    • FEN1 efficiently removes the remaining ribonucleotide after junction cleavage, consistent with a 5'-flap structure.

    Conclusions:

    • RNase HI recognizes the RNA-DNA junction on single-stranded substrates, not the heteroduplex structure.
    • This junction cleavage activity is intrinsic to RNase HI and essential for initiator RNA processing.
    • The findings support a model where Okazaki fragments are displaced, forming a 5'-flap processed sequentially by RNase HI and FEN1.