Three ribonucleases H and a reverse transcriptase from the yeast, Saccharomyces cerevisiae

Area of Science:

• Molecular Biology
• Enzymology
• Yeast Genetics

Background:

• Ribonuclease H (RNAase H) enzymes play crucial roles in nucleic acid metabolism.
• Understanding the diversity and functions of RNAase H in eukaryotic systems like yeast is essential.

Purpose of the Study:

• To isolate and characterize ribonuclease H enzymes from Saccharomyces cerevisiae.
• To investigate the enzymatic properties and potential functions of these isolated proteins, particularly RNAase H(70).

Main Methods:

• Protein isolation and purification from yeast.
• Enzymatic activity assays (ribonuclease H, reverse transcriptase, DNA polymerase stimulation).
• Immunological analysis and molecular weight determination.
• Genetic manipulation of yeast strains to study gene function.

Main Results:

• Three distinct RNAase H proteins (RNAase H(55), RNAase H(42), and RNAase H(70)) were isolated from yeast.
• RNAase H(55) and RNAase H(42) are structurally and immunologically related.
• RNAase H(70) contains both ribonuclease H and reverse transcriptase activities, with DNA polymerase stimulating activity localized to its 70 kDa polypeptide.
• Deletion mutants affecting the 160 kDa polypeptide of RNAase H(70) exhibit increased DNA content, indicating a role in DNA replication regulation.

Conclusions:

• Yeast possesses multiple RNAase H enzymes with distinct properties.
• RNAase H(70) is a multifunctional protein with potential roles in DNA replication and nucleic acid processing.
• Further investigation into the precise functions of these yeast RNAases H is warranted.