A second mitochondrial DNA primase is essential for cell growth and kinetoplast minicircle DNA replication in Trypanosoma brucei.
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.
University of California-Los Angeles, 611 Charles Young Dr. East, Los Angeles, CA 90095-1570, USA.
Jane C
JC
Hines
Dan S
DS
Ray
D000595:Amino Acid Sequence; D002460:Cell Line; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D018105:DNA, Kinetoplast / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D014353:Trypanosomiasis, African / Q000469:parasitology
D018105:DNA, Kinetoplast; D015800:Protozoan Proteins; D019915:DNA Primase
Mol Cell Biol. 2010 Mar;30(6):1319-28
20065037
J Biol Chem. 1995 Sep 8;270(36):21339-45
7545668
Mol Biochem Parasitol. 1999 Mar 15;99(1):89-101
10215027
Protist. 2001 Dec;152(4):255-62
11822657
Mol Cell Biol. 1993 Dec;13(12):7766-73
8246992
J Biol Chem. 1984 Dec 25;259(24):15532-9
6096377
PLoS Pathog. 2009 Sep;5(9):e1000589
19779567
Mol Cell. 2010 Jan 15;37(1):67-78
20129056
J Cell Biol. 1993 Dec;123(5):1069-79
8245118
Mol Cell Biol. 1996 Feb;16(2):564-76
8552084
EMBO J. 2001 Sep 3;20(17):4674-83
11532932
Mol Cell Biol. 1995 Dec;15(12):6794-803
8524245
J Biol Chem. 2006 Dec 8;281(49):37468-76
17046830
Mol Biochem Parasitol. 2001 Jun;115(1):63-7
11377740
Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7809-13
8395055
J Biol Chem. 1979 Jun 10;254(11):4895-900
438220
J Biol Chem. 1989 Jan 15;264(2):823-30
2536025
Cell. 1988 Dec 23;55(6):1083-8
2849507
Mol Biol Cell. 2003 May;14(5):1769-79
12802053
Cell. 1992 Aug 21;70(4):621-9
1324122
Mol Microbiol. 2008 Feb;67(4):820-9
18179422
J Cell Biol. 2001 May 14;153(4):735-44
11352935
Eukaryot Cell. 2002 Aug;1(4):495-502
12455998
J Cell Biol. 2001 May 14;153(4):725-34
11352934
J Biol Chem. 2003 Dec 5;278(49):49095-101
12966090
Proc Natl Acad Sci U S A. 1999 Jul 20;96(15):8455-60
10411896
J Protozool. 1977 May;24(2):325-9
881656
Nucleic Acids Res. 1998 Oct 15;26(20):4733-8
9753743
Mol Cell Biol. 1989 Jan;9(1):169-76
2927390
Eukaryot Cell. 2004 Apr;3(2):277-87
15075258
Mol Cell. 2009 Aug 28;35(4):490-501
19646907
Nucleic Acids Res. 2005 Jul 15;33(12):3875-96
16027112
Eukaryot Cell. 2006 Jan;5(1):54-61
16400168
Eukaryot Cell. 2007 Dec;6(12):2303-10
17965251
Eukaryot Cell. 2005 Apr;4(4):765-74
15821136
Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1786-9
8446592
J Biol Chem. 2002 May 10;277(19):16952-9
11859084
Mol Cell. 2002 Jul;10(1):175-86
12150917
Nucleic Acids Res. 2001 Feb 1;29(3):725-31
11160895
J Cell Biol. 1998 Nov 16;143(4):911-9
9817750
J Biol Chem. 2010 Mar 5;285(10):7056-66
20042610
Trends Parasitol. 2005 Aug;21(8):363-9
15967722
Annu Rev Biochem. 2004;73:293-320
15189144
Trends Biochem Sci. 2005 Feb;30(2):97-105
15691655
J Biol Chem. 1985 Mar 25;260(6):3844-51
3972850
Mol Cell Biol. 1989 Aug;9(8):3212-7
2552285
J Biol Chem. 1986 Feb 15;261(5):2362-8
3003111
Proc Natl Acad Sci U S A. 2004 Mar 30;101(13):4361-6
15070723
Mol Cell Biol. 2006 Jul;26(14):5382-93
16809774
Nucleic Acids Res. 1994 Nov 11;22(22):4673-80
7984417