Abstract

Dr. Champoux proposes the following studies. With respect to plus strand primer formation, it is proposed to study the cleavage specificity of RNAse H at internal sites and the influence of pre-existing nicks on subsequent cleavages. A random mutagenesis approach in MuLV will be used to define the range of sequence features of polypurine tract and its upstream region that are essential for reverse transcription. In the second area of interest, the displacement synthesis, the nature of interaction between the RT and the DNA will be probed by footprinting, photocrosslinking and use of modified DNA structures. The amino acid residues participating in melting the non-template strand during displacement synthesis will be identified by site-directed mutagenesis of the suspected residues. The energetics of the displacement process will be studied by a novel technique that relies on the measurement of positive supercoils generated during displacement synthesis.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA051605-14
Application #
6633027
Study Section
Special Emphasis Panel (ZRG1-AARR-1 (01))
Program Officer
Cole, John S
Project Start
1989-09-30
Project End
2005-04-30
Budget Start
2003-05-01
Budget End
2004-04-30
Support Year
14
Fiscal Year
2003
Total Cost
$269,622
Indirect Cost

  Institution

Name
University of Washington
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195

  Publications

Schultz, Sharon J; Zhang, Miaohua; Champoux, James J (2010) Multiple nucleotide preferences determine cleavage-site recognition by the HIV-1 and M-MuLV RNases H. J Mol Biol 397:161-78
Champoux, James J; Schultz, Sharon J (2009) Ribonuclease H: properties, substrate specificity and roles in retroviral reverse transcription. FEBS J 276:1506-16
Schultz, Sharon J; Zhang, Miaohua; Champoux, James J (2009) Preferred sequences within a defined cleavage window specify DNA 3' end-directed cleavages by retroviral RNases H. J Biol Chem 284:32225-38
Schultz, Sharon J; Champoux, James J (2008) RNase H activity: structure, specificity, and function in reverse transcription. Virus Res 134:86-103
Paulson, Benjamin A; Zhang, Miaohua; Schultz, Sharon J et al. (2007) Substitution of alanine for tyrosine-64 in the fingers subdomain of M-MuLV reverse transcriptase impairs strand displacement synthesis and blocks viral replication in vivo. Virology 366:361-76
Schultz, Sharon J; Zhang, Miaohua; Champoux, James J (2006) Sequence, distance, and accessibility are determinants of 5'-end-directed cleavages by retroviral RNases H. J Biol Chem 281:1943-55
Lanciault, Christian; Champoux, James J (2006) Pausing during reverse transcription increases the rate of retroviral recombination. J Virol 80:2483-94
Schultz, Sharon J; Zhang, Miaohua; Champoux, James J (2004) Recognition of internal cleavage sites by retroviral RNases H. J Mol Biol 344:635-52
Winshell, Jamie; Paulson, Benjamin A; Buelow, Ben D et al. (2004) Requirements for DNA unpairing during displacement synthesis by HIV-1 reverse transcriptase. J Biol Chem 279:52924-33
Lanciault, Christian; Champoux, James J (2004) Single unpaired nucleotides facilitate HIV-1 reverse transcriptase displacement synthesis through duplex RNA. J Biol Chem 279:32252-61

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