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Published on: March 18, 2015
Inhibition of reverse transcription in vivo by elevated manganese ion concentration
Eric C Bolton1, Albert S Mildvan, Jef D Boeke
1Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Abstract:
Mutations in PMR1, a yeast gene encoding a calcium/manganese exporter, dramatically decrease Ty1 retrotransposition. Ty1 cDNA is reduced in pmr1 mutant cells, despite normal levels of Ty1 RNA and proteins. The transposition defect results from Mn(2+) accumulation that inhibits reverse transcription. Cytoplasmic accumulation of Mn(2+) in pmr1 cells may directly affect reverse transcriptase (RT) activity. Trace amounts of Mn(2+) potently inhibit Ty1 RT and HIV-1 RT in vitro when the preferred cation, Mg(2+), is present. Both Mn(2+) and Mg(2+) alone activate Ty1 RT cooperatively with Hill coefficients of 2, providing kinetic evidence for a dual divalent cation requirement at the RT active site. We propose that occupancy of the B site is the major determinant of catalytic activity and that Mn(2+) at this site greatly reduces catalytic activity.
Insights
Mutations in the yeast PMR1 gene cause manganese (Mn2+) to build up, inhibiting Ty1 retrotransposition by affecting reverse transcriptase activity. This study reveals Mn2+ impairs reverse transcription, crucial for retroelement replication.
Area of Science:
- Molecular biology
- Yeast genetics
- Retrotransposition mechanisms
Background:
- The PMR1 gene in yeast encodes a calcium/manganese exporter.
- Mutations in PMR1 lead to significant reductions in Ty1 retrotransposition.
- Ty1 cDNA levels decrease in pmr1 mutants, despite normal RNA and protein expression.
Purpose of the Study:
- To investigate the molecular basis for the Ty1 retrotransposition defect in pmr1 mutant yeast.
- To determine the role of manganese (Mn2+) accumulation in inhibiting reverse transcription.
- To elucidate the effect of Mn2+ on the activity of Ty1 reverse transcriptase (RT).
Main Methods:
- Analysis of Ty1 RNA, cDNA, and protein levels in wild-type and pmr1 mutant yeast cells.
- In vitro enzymatic assays to assess the activity of purified Ty1 RT and HIV-1 RT.
- Kinetic analysis of RT activity in the presence of different divalent cations (Mn2+ and Mg2+).
Main Results:
- PMR1 mutations cause cytoplasmic Mn2+ accumulation, which inhibits Ty1 reverse transcription.
- Trace amounts of Mn2+ potently inhibit both Ty1 RT and HIV-1 RT in vitro when Mg2+ is present.
- Kinetic studies show Ty1 RT has a dual divalent cation requirement, with Mn2+ significantly reducing catalytic activity at the B site.
Conclusions:
- Manganese (Mn2+) accumulation due to PMR1 mutations is the primary cause of impaired Ty1 retrotransposition.
- Mn2+ directly inhibits reverse transcriptase activity, likely by binding to a critical site on the enzyme.
- Understanding the cation dependency of RT is crucial for retroelement and retroviral replication studies.
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