In Vivo Determination of Replication Origins of Human Mitochondrial DNA by Ligation-mediated Polymerase Chain Reaction*

(Received for publication, October 23, 1996, and in revised form, March 8, 1997)

Dongchon Kang Dagger , Kenichi Miyako , Yoichiro Kai , Takashi Irie and Koichiro Takeshige

From the Department of Biochemistry, Kyushu University School of Medicine, Fukuoka 812-82, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

A large part of replication is aborted in human mitochondria, the result being a D-loop. As few attempts have been made to distinguish free 5' ends of true replicate from those of abortive ones, we examined the 5' ends of true replicate of human mitochondrial DNA at one nucleotide resolution in vivo by making use of ligation-mediated polymerase chain reaction. The distribution and relative amounts of origins of the true replicate are exactly the same as those of total newly synthesized heavy strands, which means that the abortion of replication is independent of 5' ends. Treatment of DNA with RNase H frees 5' ends on both heavy and light strands. This is the first in vivo evidence for covalently attached primer RNA to nascent strand in human mitochondrial DNA.


INTRODUCTION

Replication of mitochondrial DNA (mtDNA) begins with synthesis of the heavy strand (H strand)1 from the replication origin OH, following transcription of the light strand (L strand). Transcription of the L strand is regulated by the L strand promoter (LSP) sequence. When synthesis of the H strand has proceeded about two-thirds, synthesis of the L strand begins from replication origin OL. Thus, the total replication rate of mtDNA is determined by H strand synthesis (for reviews, see Refs. 1-3). The multiple replication origins of human mitochondrial H and L strands have been determined as free 5' ends of mtDNA extracted from prepared mitochondria. The molecular mechanism and physiological significance of these events are poorly understood.

A large part of the synthesis of H strand is aborted, leaving a displacement loop ((D-loop) or 7 S DNA). For example, over 95% of the newly synthesized H strand is a D-loop in the case of mouse L cells (4). This makes it difficult to precisely determine origins of the true replication form (nascent H strand) (5). Few investigators have distinguished the free 5' end of the true replicate form from that of the D-loop even though selective detection of the free 5' end of the true replicate form is critical to determine replication origin. Although the 5' ends of the nascent H strand are suggested to be the same as those of D-loop in human mitochondria (5), it is uncertain whether the distribution and relative amount of the 5' ends of nascent H strand are exactly the same as those of the D-loop. In addition, the free 5' ends of human mtDNA are not set at one nucleotide resolution (6). To precisely determine the replication origin but not the simple free 5' end, it is required to selectively detect the replication origin at a higher resolution.

We made use of the ligation-mediated polymerase chain reaction (LMPCR) to detect free 5' ends (7, 8), an approach which makes feasible use of total DNA extracted directly from whole cells. By selective amplification of nascent H (not D-loop), we determined precise and comprehensive replication origins of the H strand in vivo. Here, we describe sites of free 5' ends and the transition sites of RNA to DNA for both H and L strands at one nucleotide resolution.


EXPERIMENTAL PROCEDURES

Materials

BamHI, RNase inhibitor, and T4 DNA ligase were purchased from Takara (Seta, Japan). RNase A and diethylpyrocarbonate were from Sigma. Proteinase K was from Boehringer Mannheim (Mannheim, Germany). Long Ranger® was from FMC® BioProducts (Rockland, ME). Vent DNA polymerase was from New England Biolabs (Beverly, MA). Other reagents were of analytical grade.

Preparation of DNA

HeLa MRV11 and Jurkat (human T cell leukemia line) cells were cultured as described (9) and were harvested in logarithmic proliferation phase. The cells (about 106 cells) were centrifuged, and the pellets were rapidly denatured and solubilized in 100 µl of denaturing buffer containing 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1% SDS, and 0.1 mg/ml proteinase K at 50 °C for 1 h. Total DNA was isolated by two extractions with phenol/chloroform (1/1), and then DNA was ethanol precipitated. The pellets were dried, solubilized in 100 µl of distilled water, and treated with RNase A (0.1 µg/µl) and BamHI (0.1 unit/µl). DNA was extracted with phenol/chloroform (1/1), ethanol precipitated, and solubilized in water. The amount of the total DNA was determined by measuring A260. In some cases, the initially extracted DNA was solubilized in diethylpyrocarbonate-treated water containing RNase inhibitor (0.7 units/µl) and digested with BamHI alone. After extracting DNA with phenol/chloroform (1/1), precipitating with ethanol, and solubilization in water, DNA was treated with either RNase A (0.1 µg/µl) alone or RNase A plus RNase H (6 units/µl) before LMPCR.

Preparation of Mitochondria

Mitochondria of HeLa cells were prepared by differential centrifugation (9). Briefly, the cells suspended in buffer (TES) containing 0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, and 0.1 mM EDTA were homogenized with a Potter-Elvehjem homogenizer and centrifuged at 600 × g for 10 min at 4 °C. The supernatant was centrifuged at 7,000 × g for 10 min. The pellets were washed three times with TES. The mitochondrial fraction was allowed to proceed in in vitro replication at 37 °C for 1 h, as described by Koike et al. (10, 11) prior to DNA extraction.

Ligation-mediated PCR

The primer sets used in this study are shown in Tables I and II. A unidirectional linker was prepared by hybridizing LMPR1 (5'-gcggtgacccgggagatctgtattc-3') and LMPR2 (5'-gaatacagatc-3'). DNA was chemically modified for the sequence ladder according to the method of Maxam and Gilbert (12). LMPCR was performed essentially according to the method of Mueller et al. (13) as follows.

Table I. Combination of primers for LMPCR


Set Primer 1 Primer 2 Primer 3 

D-6 D6 FD6
D-7 D7 FD7
D-8 D8 FD8
H H1 H2 FD6
D D1 D2 FD6
OL RL5837OL RFL5815OL

Table II. Primers for PCR


Primer Position on mitochondrial DNAa Length 5'-FITC Annealing

bp °C
D6 L58 -76 19  - 60
FD6 L76 -100 25 + 65
D7 L167 -186 20  - 54
FD7 L182 -207 26 + 64
D8 L312 -329 18  - 60
FD8 L322 -349 28 + 65
H1 L16036 -16055 20  - 54
H2 L16055 -16079 25  - 60
D1 L16108 -16128 21  - 54
D2 L16128 -16153 26  - 60
RL5837OL H5818 -5837 20  - 60
RFL5815OL H5791 -5815 25 + 65
RL268D6 H249 -268 20  - 54
RFL251D6 H227 -251 25 + 60

a Prescripts L and H denote L and H strands, respectively.

Primer Extension

Primer 1 was extended in 30 µl of the first strand synthesis reaction mixture consisting of 40 mM NaCl, 10 mM Tris-HCl, pH 8.9, 5 mM MgSO4, 0.01% gelatin, 0.3 pmol primer 1, 0.2 mM of each dNTP, 0.5 units of Vent DNA polymerase, and DNA (0.2 µg for sequence ladder and 0.4 µg for detection of 5' ends). DNA was denatured at 95 °C for 5 min, and the primer was annealed at 54-60 °C for 30 min, after which polymerase reaction was performed at 76 °C for 10 min.

Ligation

After cooling on ice, 45 µl of ice-cold ligation mix was added, and the ligation reaction was performed at 16 °C for more than 6 h. The ligation mix consisted of 77 mM Tris-HCl, pH 7.5, 13.3 mM MgCl2, 33.3 mM dithiothreitol, 8.3 mg/ml bovine serum albumin, 1.7 mM ATP, 100 pmol of unidirectional linker, and 3 Weiss units of T4 DNA ligase. DNA was precipitated with 9.4 µl of ice-cold precipitation mix (2.7 M sodium acetate, pH 7.0, and 1 mg of tRNA) and 220 µl of ice-cold ethanol, and then the entire mixture was kept at -20 °C for 2 h.

PCR Amplification

DNA pellets were solubilized in 66.5 µl of water, and then 33.5 µl of amplification mix (123 mM NaCl, 61.5 mM Tris-HCl, pH 8.9, 15 mM MgSO4, 0.03% gelatin, 0.3% Triton X-100, 10 pmol LMPR1, 20 pmol primer 2, 0.67 mM of each dNTP, and 1 unit of Vent DNA polymerase) was added to the DNA solution. DNA was denatured initially at 95 °C for 3 min, and then the reaction underwent 20 PCR cycles of 95 °C for 1 min, 60-65 °C for 2 min, and 76 °C for 3 min plus an extra 5 s for each cycle. Final extension was allowed to proceed at 76 °C for 10 min. Usually, the 5' end of primer 2 was fluorescein isothiocyanate-labeled, and this step was followed by the DNA extraction and analysis as described below.

To distinguish the nascent H strand from the total newly synthesized H strand, a three-primers system was adopted. H1 and H2 were used as primers 1 and 2, respectively, for the nascent H strand. For the total newly synthesized H strand, D1 and D2 were used as primers 1 and 2, respectively. Fifty µl of end-labeling mix (40 mM NaCl, 20 mM Tris-HCl, pH 8.9, 5 mM MgSO4, 0.01% gelatin, 0.1% Triton X-100, 5 pmol LMPR1, 20 pmol FD6 as primer 3, 0.2 mM of each dNTP, and 0.5 units of Vent DNA polymerase) was added to a 50-µl aliquot of each PCR mixture after amplification for end labeling. After initial denaturation at 95 °C for 3 min, 2 cycles of reaction were run at 95 °C for 1 min, at 65 °C for 2 min, and at 76 °C for 3 min, followed by a final extension of 76 °C for 5 min.

DNA Extraction and Analysis

DNA was extracted with phenol/chloroform (1/1), ethanol precipitated, dried, and finally solubilized in 10 µl of loading buffer (80% formamide, 45 mM Tris base, 45 mM boric acid, and 1 mM EDTA). After heat denaturation, 3 µl of sample was electrophoresed through a 5% Long Ranger® 7 M urea gel. The products were scanned with FluorImager SI (Molecular Dynamics, Sunnyvale, CA) and quantified with ImageQuantTM (Molecular Dynamics, Sunnyvale, CA) software.

Sequencing D-loop Region

The D-loop region was amplified by using primers D6 and RL665D9 (5'-gctaggaccaaacctatttg-3'). The PCR product was resolved on a 1% agarose gel and then extracted. The sequence was determined on a 5% Long Ranger® 7 M urea gel using an ABI PRISMTM 377 DNA sequencer by the dye terminator cycle sequencing (Perkin Elmer) method. Primer L77 (5'-acgcgatagcattgcgagac-3') was used for the L strand or RL665D9 for the H strand.


RESULTS

Origins for H Strand Replication

Free 5' ends of the H strand were determined at one nucleotide resolution. A signal at a particular site indicates that a free 5' end is located at the 3' side of the signal by one base (e.g. a signal at nucleotide 192 indicates that nucleotide 191 is a 5' end on the H strand). The free 5' ends were clustered from nucleotides 100 to 200 (Fig. 1). Although the signal at nucleotide 152 was weaker in Jurkat cells than in HeLa cells, distribution of the signals was essentially the same between the two cell lines (Fig. 1). The sequence at nucleotide 150 on the H strand is G in HeLa MRV11 cells and A in Jurkat cells. This difference might affect usage frequency of a replication origin at nucleotide 151. The major signals were grouped into 4 regions around nucleotides 110, 150, 170, and 190. The locations were essentially the same as those reported by Chang and Clayton (6).


Fig. 1. Replication origins of H strand. Free 5' ends of the H strand in HeLa MRV11 and Jurkat cells were determined by LMPCR using primer set D-6. For sequence ladder (Lanes C, G, CT, and AG) and free 5' ends (sample lane), 0.2 µg and 0.4 µg of total DNA, respectively, were used.
[View Larger Version of this Image (50K GIF file)]

These same authors (6) have reported two bands of 5' ends (around nucleotides 220 and 310) that almost correspond to the 3' ends of possible primer RNAs and another relative strong band at nucleotide 440. We did not detect the three bands by using primer sets D-7 and D-8 (results not shown).

Selective Detection of the Nascent H Strand

As a large part of the synthesis of H strand is aborted as D-loop, there is the possibility that the 5' ends described above do not reflect origins of the true replicate (nascent H strand). The 3' termini of the human D-loop have been mapped to nucleotides 16104-16106 (14). This indicates that the newly synthesized H strand exceeding this region is the nascent H strand. Hence, we amplified the nascent H strand using primers set outside of the D-loop. We designed the other primers on the inside of the D-loop to amplify the total newly synthesized H strand (i.e. D-loop and nascent H strand). Intensity of the signals for the nascent H strand was about 40% of that for the total newly synthesized H strand (results not shown). When the conditions were selected to match the apparent signal intensity between the nascent H strand and total newly synthesized H strand, the distribution and relative amounts of signals for the nascent H strand were much the same as those of the total newly synthesized H strand (Fig. 2).


Fig. 2. Selective detection of free 5' ends of nascent H strand. A, a scheme for selective detection system is shown. The bold line is mitochondrial DNA. Narrow lines are primers. Dashed lines are extended strands. Letters D and H correspond to lanes D and H in panel B. B, LMPCR was performed using primer sets H and D to detect free 5' ends of nascent H strand (lane H) and total newly synthesized H strand (lane D), respectively. For lanes D and H, 0.4 and 0.8 µg of the total DNA of HeLa MRV11 cells, respectively, were used.
[View Larger Version of this Image (15K GIF file)]

Detection of RNA Covalently Attached to the Newly Synthesized Strand

It is considered that replication of the H strand is initiated by cleavage of the L strand transcript by RNase H-like activity. In human cells, RNA covalently attached to the newly synthesized H strand is not detectable in vivo (5, 15) although such RNA is noted in mouse cells (16). We did not detect the 5' ends at areas where the 3' ends of free RNA are mapped (6). Considering the possibility that RNA is not cleaved at the sites in vivo, we treated DNA with RNase H and then performed LMPCR. This treatment led to new free 5' ends at nucleotides 297 and 302-309 (Fig. 3, A and B) but not around nucleotide 220, corresponding to conserved sequence block (CSB) I (Fig. 3A). The appearance of new 5' ends after treatment with RNase H indicates the covalent attachment of RNA to DNA. This is apparently the first demonstration of the covalent attachment of RNA to a newly synthesized H strand in human cells.


Fig. 3. Treatment of RNase H makes new free 5' ends. 1 µg of the DNA was digested with BamHI in the presence of either RNase A alone or RNase A plus RNase H and then analyzed by LMPCR. A, LMPCR was performed as described in the legend for Fig. 2. B, to determine the accurate location of the new free 5' ends on the H strand, LMPCR was performed using primer set D-7. C, to detect free 5' ends on the L strand, LMPCR was carried out using primer set OL. The signals on the right two lanes were enhanced on computer.
[View Larger Version of this Image (50K GIF file)]

The free 5' ends on the L strand were also detected at nucleotides 5772, 5775, 5776, 5778, 5779, 5780, and 5762 by LMPCR (Fig. 3C). Although covalent attachment of RNA to the L strand is noted in an in vitro system (15), such has not been demonstrated in vivo. We obtained new free 5' ends at nucleotide 5770 and to a lesser extent at 5768, 5769, and 5774 after treatment with RNase H (Fig. 3C). These new 5' ends are located at the base of the stem portion in the proposed stem-loop structure near OL (15). This is the first example of the existence of covalently attached RNA to a human nascent L strand in vivo.

DNA from Prepared Mitochondria

Chang and Clayton (6) have detected free 5' ends in the area of nucleotide 310, without using RNase H, whereas we detected 5' ends only after RNase H treatment. Because they extracted DNA from mitochondria, it may be that their free 5' ends around nucleotide 310 are the result of activation of an endogenous RNase H-like processing enzyme during mitochondria preparations. We carried out LMPCR using DNA extracted from mitochondria and detected free 5' ends around nucleotide 310 without using RNase H treatment (Fig. 4), suggesting that the primer RNA attached to DNA can be processed at these sites by an endogenous RNase H-like activity.


Fig. 4. LMPCR using DNA extracted from mitochondria. The mitochondrial fraction was prepared from 2 × 107 of HeLa MRV11 cells, and DNA was extracted as described under "Materials and Methods" (Mt). The total DNA was extracted directly from 2 × 107 whole cells (Cell). mtDNA corresponding to the total DNA from the same number of the cells was used for LMPCR. Lane D, primer set D; lane H, primer set H.
[View Larger Version of this Image (25K GIF file)]


DISCUSSION

We selectively detected free 5' ends of the true nascent H strand by LMPCR. We found no difference in the distribution and relative amounts of 5' ends between the nascent H strand and the total newly synthesized H strand. The same distribution pattern of 5' ends of nascent H strand as that of D-loop suggests that the abortion of replication is not affected by the origin.

A transcript must form a persistent RNA-DNA hybrid to prime replication. We observed new free 5' ends only near the CSB II region after RNase H treatment of DNA (Fig. 3). This suggests that the primer RNAs extend to CSB II and covalently attach to the nascent H strand. Chang and Clayton (6) have reported that the 3' termini of free RNA are mapped to three CSB regions (CSBs I-III). Our observations suggest that the RNA-DNA hybrid at CSB II is more stable than those at the other CSBs in vivo. Consistent with this, a persistent RNA-DNA hybrid is reported to be formed under an in vitro system containing the GC-rich CSB II region downstream of a promoter (17, 18).

Although we noted a small amount of covalently attached RNA to nascent H strand near the CSB II region, almost all free 5' ends were confined downstream of the CSB I or the region between nucleotides 100 and 200. These 5' ends were free of RNA. Because the 5' ends in the region of nucleotides 100 to 200 should have been primed with RNA, the RNA-DNA hybrid should extend to nucleotides 100-200 from the LSP region (around nucleotide 400) and be processed at the region. A large part of free RNA should have free 3' ends at nucleotides 100 to 200, while most of 3' ends of free RNA are clustered in three CSBs (6). The reason for the discrepancy remains to be explained.

We extensively determined the 5' ends of the true replicate of H strand by LMPCR (Fig. 5). This approach is sensitive, rapid, facilitated, and precise. Detection of a free 5' end equals to detection of the strand with a free 5' end (i.e. nascent strand), therefore, the signal intensity reflects the amount of nascent strand. Hence, it is possible to estimate the steady-state level of replication in cells using LMPCR (19).


Fig. 5. Location of free 5' ends. The sites of free 5' ends in HeLa MRV11 cell are summarized. The site of the nucleotide with the free 5' end is corrected by one base from the site where the signal located on LMPCR. A, the sequence on H strand is shown. The sequence and numbering of nucleotides are accorded to Anderson et al. (20). The actual sequence in CSB II is 5'-TTTGGGGGGGGAGGGGGG-3' in HeLa MRV11 cells. The large circles indicate major nucleotides with free 5' ends, and the small circles indicate minor nucleotides with free 5' ends. The arrows indicate nucleotides of which the 5' ends appear only after treatment with RNase H. Three CSBs are underlined. The LSP region is expressed as shadowed and underlined letters. B, the sequence on L strand is shown. The large arrow indicates major free 5' ends that appeared after RNase H treatment, and the small arrows indicate minor free 5' ends that appeared after the treatment. Other marks are the same as in panel A.
[View Larger Version of this Image (31K GIF file)]


FOOTNOTES

*   This work was supported in part by Grant-in Aid for Scientific Research on Priority Areas (08280104) from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Present address and to whom correspondence should be addressed: Dept. of Clinical Chemistry and Laboratory Medicine, Kyushu University Faculty of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-82, Japan. Fax: 81-92-633-6194.
1   The abbreviations used are: H strand, heavy strand; L strand, light strand; LSP, light strand promoter; LMPCR, ligation-mediated polymerase chain reaction; CSB, conserved sequence block; D-loop, displacement loop.

ACKNOWLEDGEMENTS

We extend special thanks to Drs. H. Sumimoto and Y. Nakabeppu, Prof. N. Hamasaki (Kyushu University), and Prof. S. Narayanan (New York Medical College), for useful comments, and M. Ohara, for editing services.


REFERENCES

  1. Clayton, D. A. (1982) Cell 28, 693-705 [Medline] [Order article via Infotrieve]
  2. Wallace, D. C. (1992) Annu. Rev. Biochem. 61, 1175-1212 [CrossRef][Medline] [Order article via Infotrieve]
  3. Clayton, D. A. (1991) Annu. Rev. Cell Biol. 7, 453-478 [CrossRef]
  4. Bogenhagen, D., and Clayton, D. A. (1978) J. Mol. Biol. 119, 49-68 [CrossRef][Medline] [Order article via Infotrieve]
  5. Tapper, D. P., and Clayton, D. A. (1981) J. Biol. Chem. 256, 5109-5115 [Abstract]
  6. Chang, D. D., and Clayton, D. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 351-355 [Abstract]
  7. Pfeifer, G. P., Steigerwald, S. D., Mueller, P. R., Wold, B., and Riggs, A. D. (1989) Science 246, 810-813 [Medline] [Order article via Infotrieve]
  8. Mueller, P. R., and Wold, B. (1989) Science 246, 780-786 [Medline] [Order article via Infotrieve]
  9. Kang, D., Nishida, J., Iyama, A., Nakabeppu, Y., Furuichi, M., Fujiwara, T., Sekiguchi, M., and Takeshige, K. (1995) J. Biol. Chem. 270, 14659-14665 [Abstract/Free Full Text]
  10. Koike, K., and Kobayashi, M. (1973) Biochim. Biophys. Acta 324, 452-460 [Medline] [Order article via Infotrieve]
  11. Koike, K., Kobayashi, M., and Fujisawa, T. (1974) Biochim. Biophys. Acta 361, 144-154 [Medline] [Order article via Infotrieve]
  12. Maxam, A. M., and Gilbert, W. (1980) Methods Enzymol. 65, 499-560 [Medline] [Order article via Infotrieve]
  13. Mueller, P. R. (1992) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seideman, J. G., Smith, J. A., and Struhl, K., eds), pp. 15.5.1-15.5.26, John Wiley & Sons, Inc., New York
  14. Doda, J. N., Wright, C. T., and Clayton, D. A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 6116-6120 [Abstract]
  15. Wong, T. W., and Clayton, D. A. (1985) Cell 42, 951-958 [Medline] [Order article via Infotrieve]
  16. Chang, D. D., Hauswirth, W. W., and Clayton, D. A. (1985) EMBO J. 4, 1559-1567 [Abstract]
  17. Xu, B., and Clayton, D. A. (1995) Mol. Cell. Biol. 15, 580-589 [Abstract]
  18. Xu, B., and Clayton, D. A. (1996) EMBO J. 15, 3135-3143 [Abstract]
  19. Miyako, K., Kai, K., Irie, T., Takeshige, K., and Kang, D. (1997) J. Biol. Chem., 9605-9608
  20. Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J., Staden, R., and Young, I. G. (1981) Nature 290, 457-465 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.