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Eukaryotic Cell, December 2008, p. 2141-2146, Vol. 7, No. 12
1535-9778/08/$08.00+0     doi:10.1128/EC.00199-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Stem-Loop Silencing Reveals that a Third Mitochondrial DNA Polymerase, POLID, Is Required for Kinetoplast DNA Replication in Trypanosomes{triangledown}

Julian Chandler, Anthula V. Vandoros, Brian Mozeleski, and Michele M. Klingbeil*

Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003

Received 18 June 2008/ Accepted 1 October 2008

Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomes, is a catenated network containing thousands of minicircles and tens of maxicircles. The topological complexity dictates some unusual features including a topoisomerase-mediated release-and-reattachment mechanism for minicircle replication and at least six mitochondrial DNA polymerases (Pols) for kDNA transactions. Previously, we identified four family A DNA Pols from Trypanosoma brucei with similarity to bacterial DNA Pol I and demonstrated that two (POLIB and POLIC) were essential for maintaining the kDNA network, while POLIA was not. Here, we used RNA interference to investigate the function of POLID in procyclic T. brucei. Stem-loop silencing of POLID resulted in growth arrest and the progressive loss of the kDNA network. Additional defects in kDNA replication included a rapid decline in minicircle and maxicircle abundance and a transient accumulation of minicircle replication intermediates before loss of the kDNA network. These results demonstrate that POLID is a third essential DNA Pol required for kDNA replication. While other eukaryotes utilize a single DNA Pol (Pol {gamma}) for replication of mitochondrial DNA, T. brucei requires at least three to maintain the complex kDNA network.

* Corresponding author. Mailing address: Department of Microbiology, University of Massachusetts, 639 North Pleasant Street, Amherst, MA 01003. Phone: (413) 577-3823. Fax: (413) 545-1578. E-mail: klingbeil{at}microbio.umass.edu

{triangledown} Published ahead of print on 10 October 2008.

Eukaryotic Cell, December 2008, p. 2141-2146, Vol. 7, No. 12
1535-9778/08/$08.00+0     doi:10.1128/EC.00199-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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