A second mitochondrial DNA primase is essential for cell growth and kinetoplast minicircle DNA replication in Trypanosoma brucei

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.

Fig. 1.

Multiple sequence alignment. Predicted kinetoplastid PRI2 proteins from Trypanosoma brucei (Tb; 927.1.4010) region 751 to 1249, Trypanosoma cruzi (Tc; 00.1047053506529.410) region 254 to 722, Trypanosoma vivax (Tvy; 486_010173) region 574 to 1063, Leishmania braziliensis (Lbr; M12_V2.0640) region 19 to 580, Leishmania major (Lmj; F23.0680) region 1 to 543, and Leishmania infantum (LinJ; 12_V3.0590) region 1 to 545 were aligned using the ClustalW progressive multiple sequence alignment algorithm (45) in MacVector 7.2 (Accelrys). Only the region of each sequence containing the conserved RNA recognition motif (RRM) and PRICT-2 domains is shown. Amino-terminal sequences are shown in Fig. S1 in the supplemental material. The RRM is indicated by an open bar above the sequence, and the PriCT-2 domain is indicated by a filled bar above the sequence. Conserved amino acid residues are shown in shaded boxes. Aspartate and histidine residues conserved in all catalytically active members of the archeoeukaryotic primase superfamily are indicated by asterisks underneath. GeneDB accession numbers for protein sequences are indicated in parentheses.

Fig. 2.

Localization of the HA-tagged PRI2 protein. T. brucei cells expressing the PRI2 protein with a C-terminal HA tag were immunostained with anti-HA monoclonal antibody (red) and counterstained with DAPI (blue) for DNA. DAPI (A), anti-HA (B), merged fluorescent (C), and phase contrast (D) images. Bar, 5 μm.

Fig. 3.

Effect of PRI2 depletion on the growth of procyclic T. brucei. Cloned procyclic T. brucei cells transfected with the PRI2 RNAi plasmid construct were incubated in culture medium containing 1 μg/ml tetracycline (+RNAi) or without tetracycline (−RNAi). The number of cells per milliliter on the y axis is the measured value times the dilution factor. The Northern blot (inset) shows the effect of RNAi (48 h) on the PRI2 mRNA level.

Fig. 4.

Loss of kDNA during PRI2 RNAi. Cells at 0 and 2 days of PRI2 RNAi were stained with DAPI and observed by fluorescence microscopy. N, nucleus; k, kinetoplast. Bar, 5 μm.

Fig. 5.

Effect of PRI2 RNAi on minicircle and maxicircle DNAs. (A) Kinetics of loss of total minicircles and maxicircles during PRI2 RNAi. After digestion with HindIII and XbaI, total cellular DNA (2 × 10⁶ cell equivalents per lane) was fractionated on a 1.5% agarose gel containing 1 μg/ml ethidium bromide. Southern blots were probed for minicircle DNA, a maxicircle fragment (Maxi), and a hexose transporter fragment as a loading control (Load). A 1.0-kb minicircle fragment (Mini) represents the full-length minicircle. (B) Phosphorimager quantitation of the relative levels of maxicircle and minicircle DNAs shown in panel A. (C) Loss of N/C and CC maxicircles during RNAi. Southern blot of topoisomerase II-treated total DNA (2 × 10⁶ cell equivalents/lane) during PRI2 RNAi. The gel was run, Southern blotted, and probed with a maxicircle DNA probe (C) and then stripped and reprobed with the THT sequence as a chromosomal DNA loading control (D). (E) Phosphorimager quantitation of the levels of N/G (open circles) and CC (filled circles) maxicircles relative to the levels at day 0. Each lane was normalized relative to the loading control. Error bars show the standard errors of the means.

Fig. 6.

Effect of PRI2 RNAi on free minicircle replication intermediates. (A) Free minicircle DNA was purified after various times of RNAi and then fractionated (5 × 10⁵ cell equivalents per lane) on an agarose gel. Minicircle DNA was detected by Southern blotting using a minicircle probe. Intact kDNA does not enter the gel and remains in the well. The band near the top of the gel (indicated as “C”) is due to chromosomal DNA. Covalently closed (CC), nicked/gapped (N/G), and catenated minicircles (cm). (B) The blot shown in panel A was stripped and reprobed for the THT sequence as a chromosomal DNA loading control. (C) Phosphorimager quantitation of the relative levels of free minicircle species after various times of RNAi. Each lane was normalized relative to the loading control. Error bars shown the standard errors of the means.

Fig. 7.

Coelution of His₁₀-tagged PRI2ΔNT and PRI2ΔNTCS with DNA primase activity from a heparin Sepharose column after purification by metal chelate chromatography. (A) His₁₀-tagged PRI2ΔNT was expressed in E. coli BL21, purified from the cell lysate on a His-Bind column (Novagen), desalted by passage over a G-25 Sephadex column, and purified further on a heparin Sepharose column. Fractions were analyzed by electrophoresis on a 7% SDS-polyacrylamide gel and stained with Coomassie blue. (B) His₁₀-tagged PRI2ΔNTCS was expressed, purified, and chromatographed as described in the legend to in panel A. (C) Fractions obtained from panels A and B were desalted, and 15 μl of each was assayed for primase activity in a coupled reaction with Klenow DNA polymerase, 1 mM ATP, and [α-³²P]dATP on a poly(dT) template. Filled circles, PRI2ΔNT; filled squares, PRI2ΔNTCS mutant. (D) DNA binding by PRI2ΔNT and PRI2ΔNTCS. Increasing amounts of PRI2ΔNT and PRI2ΔNTCS were incubated with the ³²P-end-labeled oligonucleotide Q82 and electrophoresed on a horizontal submerged 6% polyacrylamide gel. The gel was dried and exposed to autoradiographic film.

Fig. 8.

Activity of recombinant PRI2ΔNT protein. (A) ATP-dependent synthesis of poly(dA). Fraction 15 from the heparin column was assayed for primase activity in the presence of various levels of ATP. Each reaction mixture contained 1 μg of recombinant PRI2ΔNT, poly(dT), and ATP, as indicated. Reaction mixtures were incubated for 40 min at 30°C, followed by the addition of 1 unit of Klenow DNA polymerase and 16 μM [α-³²P]dATP (24,400 cpm/pmol). Incorporated radioactivity was determined after an additional 40 min at 30°C. (B) Effect of preincubation of the poly(dT) template with PRI2ΔNT and 1 mM ATP. Filled circles, preincubation for 40 min prior to adding Klenow DNA polymerase and [α-³²P]dATP; filled squares, coupled reaction without preincubation. (C) Effect of RNaseH on priming of poly(dA) synthesis. Poly(dA) synthesis in a reaction with 40 min of incubation with PRI2ΔNT, ATP, and poly(dT), followed by 20 min at 75°C and reannealing, 30 min with (filled squares) or without (filled circles) 2.5 units of RNaseH at 37°C, 20 min at 65°C, and reannealing, and finally, 40 min at 30°C with Klenow polymerase and [α-³²P]dATP.

Fig. 9.

Elongation of poly(A) primers by Klenow DNA polymerase. To analyze the PRI2ΔNT products, a reaction mixture (175 μl) contained poly(dT) template, PRI2ΔNT (7.3 μg), and 100 μM [α-³²P]ATP (18,000 cpm/pmol) as a substrate. After incubation at 30°C for the indicated times, the reaction mixtures were processed as described in Materials and Methods. Lanes 1 to 3, incubation with PRI2ΔNT for 40, 80, and 120 min, respectively; lanes 4 to 6, equal volumes of each of samples 1 to 3 were incubated for an additional 40 min at 30°C with Klenow DNA polymerase (1.5 units) and dATP, respectively; lane 7, an equal volume of sample 3 was incubated with RNaseH (1.5 units) for 20 min at 37°C; lane O, ³²P-labeled oligonucleotide markers of indicated lengths; lane M, ³²P-labeled DNA markers of indicated lengths.