A Second Mitochondrial DNA Primase Is Essential for Cell Growth and Kinetoplast Minicircle DNA Replication in Trypanosoma brucei

Jane C Hines,Dan S Ray

doi:10.1128/ec.00308-10

Abstract

The mitochondrial DNA of trypanosomes contains two types of circular DNAs, minicircles and maxicircles. Both minicircles and maxicircles replicate from specific replication origins by unidirectional theta-type intermediates. Initiation of the minicircle leading strand and also that of at least the first Okazaki fragment involve RNA priming. The Trypanosoma brucei genome encodes two mitochondrial DNA primases, PRI1 and PRI2, related to the primases of eukaryotic nucleocytoplasmic large DNA viruses. These primases are members of the archeoeukaryotic primase superfamily, and each of them contain an RNA recognition motif and a PriCT-2 motif. In Leishmania species, PRI2 proteins are approximately 61 to 66 kDa in size, whereas in Trypanosoma species, PRI2 proteins have additional long amino-terminal extensions. RNA interference (RNAi) of T. brucei PRI2 resulted in the loss of kinetoplast DNA and accumulation of covalently closed free minicircles. Recombinant PRI2 lacking this extension (PRI2ΔNT) primes poly(dA) synthesis on a poly(dT) template in an ATP-dependent manner. Mutation of two conserved aspartate residues (PRI2ΔNTCS) resulted in loss of enzymatic activity but not loss of DNA binding. We propose that PRI2 is directly involved in initiating kinetoplast minicircle replication.

Highlights

The mitochondrial DNA of trypanosomes has long been known to have a novel network structure consisting of topologically interlocked minicircles and maxicircles [30]
Minicircles replicate by a unidirectional theta-type intermediate from an origin sequence that is specified by the universal minicircle sequence (UMS) [1, 2, 33, 34, 46]
We recently identified a mitochondrial DNA primase (PRI1) in T. brucei that is essential for cell growth and kDNA replication [16]

Summary

Introduction

The mitochondrial DNA (kinetoplast DNA [kDNA]) of trypanosomes has long been known to have a novel network structure consisting of topologically interlocked minicircles and maxicircles [30]. Minicircles encode small RNA species termed guide RNAs, which serve as templates in the insertion or deletion of U residues in the maxicircle transcripts by a mechanism termed RNA editing [44] Studies of this unusual form of mitochondrial DNA focused on the mechanisms of replication of the DNA using light and electron microscopies, radioactive labeling, sedimentation, gel electrophoresis, and restriction enzyme analysis. Minicircle replication intermediates and other replication proteins were subsequently localized to the antipodal sites [12, 18] These include a ␤-type DNA polymerase [38], a structure-specific endonuclease (SSE1) [8, 10], a DNA ligase (ligase k␤) [42], RNase H [9], two DNA helicases (PIF1 and PIF5) [28, 29], a H1 histone-like protein (KAP4) [49], a minicircle origin binding protein (p38) [26], a type IA topoisomerase [39], and a DNA primase (PRI1) [16].

Methods

Results

Conclusion