Open AccessA Mitochondrial DNA Primase Is Essential for Cell Growth and Kinetoplast DNA Replication in Trypanosoma brucei
Author(s) -
Jane C. Hines,
Dan S. Ray
Publication year - 2010
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.01231-09
Subject(s) - kinetoplast , biology , minicircle , dna replication , primase , dna polymerase , okazaki fragments , trypanosoma brucei , microbiology and biotechnology , dna clamp , dna polymerase ii , dna , prokaryotic dna replication , eukaryotic dna replication , biochemistry , gene , rna , reverse transcriptase
Kinetoplast DNA in African trypanosomes contains a novel form of mitochondrial DNA consisting of thousands of minicircles and dozens of maxicircles topologically interlocked to form a two-dimensional sheet. The replication of this unusual form of mitochondrial DNA has been studied for more than 30 years, and although a large number of kinetoplast replication genes and proteins have been identified,in vitro replication of these DNAs has not been possible since a kinetoplast DNA primase has not been available. We describe here aTrypanosoma brucei DNA primase gene,PRI1 , that encodes a 70-kDa protein that localizes to the kinetoplast and is essential for both cell growth and kinetoplast DNA replication. The expression ofPRI1 mRNA is cyclic and reaches maximum levels at a time corresponding to duplication of the kinetoplast DNA. A 3′-hydroxyl-terminated oligoriboadenylate is synthesized on a poly(dT) template by a recombinant form of the PRI1 protein and is subsequently elongated by DNA polymerase and added dATP. Poly(dA) synthesis is dependent on both PRI1 protein and ATP and is inhibited by RNase H treatment of the product of PRI1 synthesis.