RESUMO
The mitochondrial DNA (kinetoplast DNA) in trypanosomatids exists as a highly organized nucleoprotein structure with the DNA consisting of thousands of interlocked circles. Four H1 histone-like proteins (KAP1, 2, 3 and 4) are associated with the kinetoplast DNA in the trypanosomatid Crithidia fasciculata. We have disrupted both alleles of the KAP1 gene in this diploid protozoan and shown that expression of the KAP1 protein is eliminated. The mutant strain is viable but has substantial rearrangement of the kinetoplast structure. Expression of the KAP1 protein from an episome restored expression of the KAP1 protein in the mutant strain and also restored a normal kinetoplast structure. These studies provide evidence that the KAP1 protein is involved in kinetoplast DNA organization in vivo but is nonessential for cell viability.
Assuntos
Crithidia fasciculata/ultraestrutura , DNA de Cinetoplasto/ultraestrutura , Proteínas de Ligação a DNA/genética , Deleção de Genes , Proteínas de Protozoários , Alelos , Animais , Crithidia fasciculata/genética , Crithidia fasciculata/crescimento & desenvolvimento , DNA de Cinetoplasto/genética , Proteínas de Ligação a DNA/metabolismo , Diploide , Genes de Protozoários , Microscopia Eletrônica , PlasmídeosRESUMO
It has been shown in vitro that Saccharomyces cerevisiae strand exchange protein 1 (Sep1) promotes the transfer of one strand of a linear duplex DNA to a homologous single-stranded DNA circle. Sep1 also has an exonuclease active on DNA and RNA. By using exonuclease III-treated linear duplex DNA with various lengths of single-stranded tail as well as Ca2+ to inhibit the exonuclease activity of Sep1, we show that the processivity of exonuclease activity of Sep1 is greater than previously reported. The results in this work also demonstrate that the joint molecule between the linear duplex and single-stranded circle observed from the Sep1-promoted strand transfer reaction is just the pairing between the long single-stranded tail of the linear duplex DNA (generated by the exonuclease activity of Sep1) and the single-stranded circular DNA. When a synthetic Holliday junction was used as substrate, branch migration facilitated by Sep1 could not be detected. Finally, using electron microscopy no alpha-structure, a joint molecule with displaced single-stranded DNA tail that indicates branch migration could be observed. The results imply that Sep1 cannot promote branch migration in vitro. Further investigation is needed to determine the role of Sep1 in recombination in vivo.
