Abstract

The presence of a curved DNA sequence in the gene for the NADH-dehydrogenase subunit 2 of rat mitochondrial genome, upstream from the origin of the light strand replication have been demonstrated through theoretical analysis and experimental approaches. Gel retardation assays showed that this structure makes a complex with a protein component extracted from the mitochondrial matrix. The isolation and purification of this protein is reported. With a Sepharose CL-6B and magnetic DNA affinity chromatography a polypeptide was purified to homogeneity having 25-kDa mass as shown by gel electrophoresis. To functionally characterize this protein, its capability to bind to other sequences of the homologous or heterologous DNA and to specific riboprobes was also investigated. A role for this protein as a trans-acting agent required for the expression of the mammalian mitochondrial genome is suggested.

Highlights

  • Mammalian mtDNAs1 have two separate and distinct replication origins

  • This structure acts as a binding site for a nuclearly coded 67-kDa polypeptide isolated from the matrix and we suggested that it is a regulatory factor promoting the first step of the H strand replication through the RNA 3 DNA transition [12, 13]

  • In this paper we report the localization of a curved DNA sequence inside the gene for the NADH dehydrogenase subunit 2 (ND2) upstream Ori-L

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Summary

Introduction

Mammalian mtDNAs1 have two separate and distinct replication origins. The origin of the heavy strand replication (OriH), inside the so-called D-loop containing region which contains the promoters for the transcription of both strands (H and L strand promoters); and the origin of the L strand synthesis (Ori-L), nested within a cluster of five tRNA genes at two-thirds of the molecule, clockwise with respect to the Ori-H (for review, see Ref. 1). We have already reported the presence of a curved DNA in the D-loop containing region, located close to Ori-H This structure acts as a binding site for a nuclearly coded 67-kDa polypeptide isolated from the matrix and we suggested that it is a regulatory factor promoting the first step of the H strand replication through the RNA 3 DNA transition [12, 13]. We present the purification to homogeneity and characterization of a 25-kDa protein unknown so far, able to bind this DNA structure This is one of the few proteins, nuclearly coded, purified until now from higher eukaryotes which should participate in the biogenesis of mitochondria and for which we suggest a possible physiological role

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