Abstract

The long term objectives of this project are to investigate the structure and mechanism of replication of kinetoplast DNA (kDNA) in trypanosomatids. Kinetoplast DNA is a network containing thousands of topologically interlocked DNA minicircles. Minicircle replication occurs after release of the circles from the network. The following projects will be initiated. First, minicircle replication will be established in vitro. The substrate will be a plasmid containing a minicircle replication origin, and proteins will be provided by a mitochondrial extract. Second, proteins involved in kDNA replication will be purified and studied. Purification will rely heavily on affinity techniques using monoclonal antibodies, and these antibodies will also be used to immunolocalize the proteins and to study their mechanism of action. Third, using the in vitro system and purified proteins, minicircle sequences important to replication will be studied. These experiments will involve in vitro replication or binding of proteins with normal or mutated minicircle sequences. Fourth, topology of kDNA networks will be investigated, in hopes of determining their pattern of organization. These studies will involve characterization of minicircle oligomers released from networks by random minicircle cleavage. Finally, the structure of networks in vivo will be investigated using high resolution fluorescence microscopic techniques that allow three dimensional image reconstruction. These studies should allow determination of changes in network structure which occur during replication, and, using in situ hybridization, the localization of the minicircle bent helix. Overall, these studies should provide basic knowledge of DNA structure and replication; in addition, since humans contain no DNA resembling a kDNA network, they may suggest novel modes of anti-parasite chemotherapy.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM027608-25
Application #
3484617
Study Section
Biochemistry Study Section (BIO)
Project Start
1980-01-01
Project End
1994-12-31
Budget Start
1993-01-01
Budget End
1993-12-31
Support Year
25
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218

  Publications

Motyka, Shawn A; Zhao, Zhixing; Gull, Keith et al. (2004) Integration of pZJM library plasmids into unexpected locations in the Trypanosoma brucei genome. Mol Biochem Parasitol 134:163-7
Saxowsky, Tina T; Choudhary, Gunjan; Klingbeil, Michele M et al. (2003) Trypanosoma brucei has two distinct mitochondrial DNA polymerase beta enzymes. J Biol Chem 278:49095-101
Saxowsky, Tina T; Matsumoto, Yoshihiro; Englund, Paul T (2002) The mitochondrial DNA polymerase beta from Crithidia fasciculata has 5'-deoxyribose phosphate (dRP) lyase activity but is deficient in the release of dRP. J Biol Chem 277:37201-6
Grams, Jayleen; Morris, James C; Drew, Mark E et al. (2002) A trypanosome mitochondrial RNA polymerase is required for transcription and replication. J Biol Chem 277:16952-9
Lukes, Julius; Guilbride, D Lys; Votypka, Jan et al. (2002) Kinetoplast DNA network: evolution of an improbable structure. Eukaryot Cell 1:495-502
Wang, Zefeng; Drew, Mark E; Morris, James C et al. (2002) Asymmetrical division of the kinetoplast DNA network of the trypanosome. EMBO J 21:4998-5005
Klingbeil, Michele M; Motyka, Shawn A; Englund, Paul T (2002) Multiple mitochondrial DNA polymerases in Trypanosoma brucei. Mol Cell 10:175-86
Morris, J C; Wang, Z; Drew, M E et al. (2001) Inhibition of bloodstream form Trypanosoma brucei gene expression by RNA interference using the pZJM dual T7 vector. Mol Biochem Parasitol 117:111-3
Morris, J C; Drew, M E; Klingbeil, M M et al. (2001) Replication of kinetoplast DNA: an update for the new millennium. Int J Parasitol 31:453-8
Klingbeil, M M; Drew, M E; Liu, Y et al. (2001) Unlocking the secrets of trypanosome kinetoplast DNA network replication. Protist 152:255-62

Showing the most recent 10 out of 48 publications