Linearized free maxicircle DNA in Crithidia fasciculata is a product of topoisomerase II-mediated cleavage.

Abstract:

:Linearized free maxicircle DNA, present in detergent lysates of Crithidia fasciculata mitochondria, was thought to be a replication intermediate formed during rolling circle replication of maxicircle DNA. Gel electrophoresis of the linearized free maxicircles indicated that they were slightly larger than the maxicircle genome, raising the possibility of the presence of terminal repetitions (Hajduk, S.L., Klein, V.A. and Englund, P.T. (1984) Cell 36, 483-492). We recently found, however, that maxicircles replicate by a theta-mechanism, and not as rolling circles (Carpenter, L.R. and Englund, P.T. (1995) Mol. Cell Biol. 15, 6794-6803). Given that theta-replication does not easily explain the presence of linearized free maxicircles, we investigated alternative explanations for their existence. We present evidence that this DNA species results from the double-strand cleavage of maxicircles due to detergent denaturation of intracellular topoisomerase II cleavable complexes. As expected for a topoisomerase II cleavage product, the linearized free maxicircle DNA is covalently bound to protein at both 5' ends. In addition, the slightly larger apparent size of linearized free maxicircle DNA or maxicircles linearized by a restriction enzyme can be explained by anomalous electrophoretic migration during conventional or pulsed-field agarose gel electrophoresis. This anomalous migration is presumably due to bends or other unusual structures in the DNA.

journal_name

Mol Biochem Parasitol

authors

Carpenter LR,Englund PT

doi

10.1016/0166-6851(95)02550-2

subject

Has Abstract

pub_date

1996-02-01 00:00:00

pages

115-23

issue

1-2

eissn

0166-6851

issn

1872-9428

pii

0166-6851(95)02550-2

journal_volume

76

pub_type

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