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. 2004 Jun 16;23(12):2423-9.
doi: 10.1038/sj.emboj.7600257. Epub 2004 May 27.

Reconstitution of a minimal mtDNA replisome in vitro

Jenny A Korhonen  1 , Xuan Hoi Pham, Mina Pellegrini, Maria Falkenberg
Affiliations

Reconstitution of a minimal mtDNA replisome in vitro

Jenny A Korhonen et al. EMBO J. .

Abstract

We here reconstitute a minimal mammalian mitochondrial DNA (mtDNA) replisome in vitro. The mtDNA polymerase (POLgamma) cannot use double-stranded DNA (dsDNA) as template for DNA synthesis. Similarly, the TWINKLE DNA helicase is unable to unwind longer stretches of dsDNA. In combination, POLgamma and TWINKLE form a processive replication machinery, which can use dsDNA as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. The addition of the mitochondrial ssDNA-binding protein stimulates the reaction further, generating DNA products of about 16 kb, the size of the mammalian mtDNA molecule. The observed DNA synthesis rate is 180 base pairs (bp)/min, corresponding closely to the previously calculated value of 270 bp/min for in vivo DNA replication. Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. Furthermore, mutations in TWINKLE and POLgamma cause autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with deletions in mitochondrial DNA. The functional interactions between TWINKLE and POLgamma thus explain why mutations in these two proteins cause an identical syndrome.

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Figures

Figure 1
Figure 1
POLγ cannot use dsDNA as template for DNA synthesis. (A) The mini-circle template was prepared as described in Materials and methods. DNA synthesis is initiated at the 3′-hydroxy terminus and proceeds 20 nt before it encounters the dsDNA region of the template. (B) Recombinant POLγ (1 μg) purified over heparin sepharose was separated by SDS–PAGE (12.5%) and revealed with Coomassie brilliant blue staining. (C) POLγ (300 fmol) was incubated together with the mini-circle template at 37°C in a total reaction mixture of 75 μl as described in Materials and methods. At times indicated, 10 μl was removed and the reaction was terminated by addition of 10 μl of gel-loading buffer (98% formamide, 10 mM EDTA (pH 8.0), 0.025% xylene cyanol FF, and 0.025% bromophenol blue). The samples were analyzed on a 10% denaturing polyacrylamide gel.
Figure 2
Figure 2
TWINKLE cannot unwind longer stretches of double-stranded DNA, even in the presence of mtSSB. (A) TWINKLE was added to a reaction mixture containing the short 20-bp double-stranded template as described in Materials and methods, and incubated for 30 min. Lane 1, untreated substrate; lane 2, substrate heated to 100°C before loading; lane 3, 550 fmol Twinkle; S, double-stranded substrate; P, single-stranded product. (B) TWINKLE is unable to unwind a 55-bp double-stranded template. Lane 3, 550 fmol Twinkle; lane 4, 550 fmol TWINKLE and 5 pmol mtSSB; lane 5, 5 pmol mtSSB; lane 6, 550 fmol UL5/52/8; lane 7, 550 fmol UL5/52/8 and 5 pmol ICP8; lane 8, 5 pmol ICP8.
Figure 3
Figure 3
Rolling-circle DNA replication. (A) The mini-circle template was prepared as described in Materials and methods, and [α-32P]dCTP was used to preferentially label the leading strand. (B) TWINKLE alone cannot unwind the mini-circle template. TWINKLE was added to a reaction mixture containing the mini-circle template as described in Materials and methods, and incubated for 30 min. Lane 1, untreated substrate; lane 2, substrate heated to 100°C before loading; lane 3, 550 fmol TWINKLE; S, double-stranded substrate; P, single-stranded product. (C) Increasing amounts of TWINKLE were added to the mini-circle template together with 100 fmol POLγ as described under Materials and methods, and then analyzed on a 0.8% denaturating agarose gel. Lane 1, size marker; lane 2, 1.65 pmol TWINKLE (hexamer); lane 3, 0 fmol TWINKLE; lane 4, 55 fmol TWINKLE; lane 5, 180 fmol TWINKLE; lane 6, 550 fmol TWINKLE; lane 7, 1.65 pmol Twinkle. (D) A constant amount of TWINKLE (550 fmol, hexamer) and POLγ (100 fmol) was added when indicated, together with an increasing amount of mtSSB and analyzed as above. Lane 1, size marker; lane 3, 0 pmol mtSSB; lane 4, 0.1 pmol mtSSB; lane 5, 0.5 pmol mtSSB; lane 6, 1.0 pmol mtSSB; lane 7, 5 pmol mtSSB; lane 8, 10 pmol mtSSB; lane 9, 0 pmol mtSSB; lane 10, 0.1 pmol mtSSB; lane 11, 0.5 pmol mtSSB; lane 12, 1.0 pmol mtSSB; lane 13, 5 pmol mtSSB; lane 14, 10 pmol mtSSB.
Figure 4
Figure 4
The mitochondrial replication machinery can utilize both ATP and GTP as energy sources. TWINKLE (0.55 pmol), DNA POLγ (100 fmol), and mtSSB (5 pmol) were added to the reaction, together with 4 mM of the indicated NTP. In lane 2, we added 1 mM of each NTP. The reactions were incubated at 37°C for 60 min, and treated as described in Materials and methods. The reactions were analyzed on a 0.8% denaturating agarose gel.
Figure 5
Figure 5
(A) The UL5/8/52 DNA helicase cannot replace TWINKLE at the mtDNA replication fork. We added POLγ (100 fmol), TWINKLE (0.9 pmol), mtSSB (5 pmol), and UL5/52/8 (0.9 pmol). The reactions were incubated for 60 min at 37°C and treated as described under Materials and methods. (B) The T4 and T7 DNA polymerases cannot replace POLγ at the mtDNA replication fork. The replication reactions were as described in (A), with T4 pol (1 U), T7 (1 U), and T4 gp32 (5 pmol). Reactions were performed with 4 mM ATP, but identical results were obtained with GTP as cofactor (data not shown).
Figure 6
Figure 6
The mitochondrial DNA replication machinery can synthesize ssDNA at a rate of 180 bp/min on the dsDNA mini-circle template. (A) We incubated TWINKLE (2.7 pmol), DNA POLγ (500 fmol, mtSSB (25 pmol), and the mini-circle template (175 fmol) at 37°C in a total reaction volume of 125 μl as described in Materials and methods. At times indicated, we removed 12.5 μl and the reaction was stopped as described in Materials and methods. The reactions were analyzed on a 0.8% denaturating agarose gel. (B) A schematic representation of the single-stranded M13 mp18 DNA template used to measure DNA synthesis rate. (C) The DNA synthesis rate of POLγ on single-stranded M13 mp18 DNA in the presence of mtSSB was about 350 nt/min. DNA POLγ (500 fmol) and 25 pmol mtSSB were incubated together with a primed M13 ssDNA at 37°C in a total reaction mixture of 125 μl as described in Materials and methods. At times indicated, 12.5 μl was removed and the reaction was terminated by addition of 3-μl of stop solution. The reactions were analyzed by nondenaturing agarose gel electrophoresis. (D) The rate of T7 DNA polymerase together with mtSSB on ssDNA was 1500 nt/min. The reactions were as described in (C), but T7 DNA polymerase (5 U) was used instead of POLγ.
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References

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