(Received for publication, October 23, 1996, and in revised form, March 8, 1997)
From the Department of Biochemistry, Kyushu University School of Medicine, Fukuoka 812-82, Japan
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.
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.
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.
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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.
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.
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 AnalysisDNA 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 RegionThe 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.
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).
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).
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).
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.
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.
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.
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).
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.