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Identification of mutant adeno-associated virus Rep proteins which are dominant-negative for DNA helicase activity
1Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA, 20892.
Abstract:
Adeno-associated virus type 2 (AAV) Rep proteins have been postulated to play a role in unwinding the 145-bp inverted terminal repeats during AAV DNA replication. Previous studies showed that AAV Rep78 and Rep68 could unwind a DNA partial duplex of 26 bp. In this work it is demonstrated that nuclear extracts of human 293 cells containing wild-type Rep68 can unwind partial DNA duplexes up to 160 bp long. Mutant Rep proteins with either a histidine substituted for lysine 340 or a deletion of methionine 225 had no detectable helicase activity and inhibited the helicase activity of wild-type Rep68 protein. This observation is consistent with the model that the functional form of the Rep proteins is a multimer.
Insights
Adeno-associated virus type 2 Rep proteins are crucial for unwinding DNA during replication. Mutant proteins lacking helicase activity suggest Rep proteins function as multimers.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Adeno-associated virus type 2 (AAV) Rep proteins are essential for viral DNA replication.
- Previous research indicated AAV Rep78 and Rep68 proteins could unwind short DNA duplexes (26 bp).
Purpose of the Study:
- To investigate the DNA unwinding capabilities of wild-type and mutant AAV Rep68 proteins.
- To explore the role of specific amino acid residues and protein multimerization in Rep protein function.
Main Methods:
- Utilized nuclear extracts from human 293 cells expressing wild-type and mutant Rep68 proteins.
- Assessed DNA helicase activity by measuring the unwinding of partial DNA duplexes of varying lengths (up to 160 bp).
Main Results:
- Wild-type Rep68 demonstrated the ability to unwind DNA partial duplexes up to 160 bp.
- Mutant Rep proteins (H340K substitution or M225 deletion) exhibited no helicase activity.
- These mutant proteins also inhibited the helicase activity of wild-type Rep68.
Conclusions:
- AAV Rep68 possesses significant DNA unwinding activity, extending beyond previously reported lengths.
- The loss of helicase activity in mutant proteins supports the hypothesis that Rep proteins function as multimers for DNA replication.
- These findings provide insights into the mechanism of AAV DNA replication and the role of Rep proteins.