(Received for publication, April 26, 1995; and in revised form, July 6, 1995)
From the
Using a stringent purification procedure on single-stranded DNA cellulose, we have isolated the mitochondrial single-stranded DNA-binding protein from Drosophila melanogaster embryos. Its identity is demonstrated by amino-terminal sequencing of the homogeneous protein and by its localization to a mitochondrial protein fraction. The mitochondrial protein is immunologically and biochemically distinct from the previously characterized nuclear replication protein A from Drosophila (Mitsis, P. G., Kowalczykowski, S. C., and Lehman, I. R.(1993) Biochemistry 32, 5257-5266; Marton, R. F., Thömmes, P., and Cotterill, S.(1994) FEBS Lett. 342, 139-144). It consists of a single polypeptide of 18 kDa, which is responsible for the DNA binding activity. Sedimentation analysis suggests that D. melanogaster mitochondrial single-stranded DNA-binding protein exists as a homo-oligomer, possibly a tetramer, in solution. The protein binds to DNA in its single-stranded form with a strong preference over double-stranded DNA or RNA, and binds to polypyrimidines preferentially over polypurines. Drosophila mitochondrial single-stranded DNA-binding protein exhibits a greater affinity for long oligonucleotides as compared to short ones, yet does not show high cooperativity. Its binding site size, determined by competition studies and by fluorescence quenching, is approximately 17 nucleotides under low salt conditions, and increases in the presence of greater than 150 mM NaCl. The homogeneous protein stimulates the activity of mitochondrial DNA polymerase from D. melanogaster embryos, increasing dramatically the rate of initiation of DNA synthesis on a singly primed DNA template.
Many of the processes involved in DNA metabolism including DNA
replication, recombination, and repair, generate intermediates
containing single-stranded regions of DNA. These regions are stabilized
and kept accessible for the various catalytic processes by the binding
of single-stranded DNA-binding proteins (SSBs). ()Prokaryotic SSBs (e.g.Escherichia coli (Eco) SSB and bacteriophage T4 gene 32 protein) are
generally small proteins which bind to single-stranded DNA (ssDNA) with
high affinity. They show high specificity for ssDNA over
double-stranded DNA (dsDNA) and RNA, but display little sequence
specificity (reviewed in (1, 2, 3) ).
Although they do not exhibit direct catalytic function, they stimulate
DNA replication in vitro.
Mitochondrial DNA replication is
independent from chromosomal DNA replication and is carried out largely
with specific mitochondrial replication proteins including the
mitochondrial DNA polymerase (pol ) and an SSB (mtSSB) distinct
from the nuclear SSB, replication protein A (RP-A). mtSSB appears to
serve an important function during mtDNA replication, by stabilizing
the displaced ssDNA that is the template for lagging DNA strand
synthesis(4) . mtSSBs have been isolated from several species
including rat(4, 5) , Xenopus
laevis(6) , and yeast(7) . These proteins consist
of a single small (13-16 kDa) polypeptide, which shows a high
degree of similarity to Eco SSB in its primary
structure(7, 8) . Although all the functions of mtSSB
in mtDNA metabolism have not been defined, it is critical for
replication, because deletion of the yeast protein (RIM1) causes loss
of mitochondrial DNA(7) . Consistent with a role in mtDNA
replication, interactions between mtSSB and other mitochondrial
replication proteins have been observed. In vitro studies
indicate that under some conditions, the rat and X. laevis mtSSBs stimulate partially purified forms of mitochondrial DNA
polymerase(9, 10) , and a putative human mtSSB
stimulates human pol
(11) . In addition, genetic evidence
from yeast suggests an interaction between RIM1 and the mtDNA helicase,
PIF1(7) .
We have purified a single-stranded DNA-binding protein from Drosophila embryos (hereafter called Dm mtSSB) to near homogeneity. Its physical and biochemical properties demonstrate that it is distinct from the nuclear SSB, dRP-A, but has a high degree of similarity to Eco SSB and to eukaryotic mtSSBs. Further, its functional interaction with the near-homogeneous mitochondrial DNA polymerase from Drosophila melanogaster embryos (12) suggests that it serves an important role in Drosophila mtDNA replication.
To assess the affinity of binding of the proteins to various oligo- and polynucleotides, a competitive gel mobility shift assay was used. Here, the labeled oligonucleotide and the unlabeled competitor were mixed prior to addition of the protein. The amount of competitor added was calculated as mol of nucleotide.
In the analysis of pol processivity, reactions were as
described above, except that reaction mixtures contained 30 µM dATP, 30 µM dCTP, 30 µM dGTP, 10
µM [
-
P]dTTP (2
10
cpm/pmol), 20 µM (as nucleotides) singly
primed wild-type M13 DNA, and 0.02 unit of Fraction VI enzyme (100-fold
excess of primer ends over pol
molecules). Reaction products were
isolated and analyzed by gel electrophoresis as described(16) .
In the analysis of limited primer extension by pol , reaction
mixtures (0.05 ml) contained 50 mM Tris
HCl (pH 8.5), 4
mM MgCl
, 10 mM dithiothreitol, 30
mM KCl, 400 µg/ml bovine serum albumin, 30 µM
dATP, 30 µM dGTP, 30 µM dTTP, 20 µM (as nucleotides) 5`-end-labeled, singly primed recombinant M13
DNA, and 0.4 unit of Fraction VI enzyme (3-fold excess of primer ends
over pol
molecules). Incubation was at 30 °C for 5 min.
Reaction products were isolated and analyzed by gel electrophoresis as
previously described(16) .
Figure 1: Purification of Dm mtSSB from early embryos. Aliquots of protein fractions derived upon the purification of Dm mtSSB were analyzed by 15% SDS-polyacrylamide gel electrophoresis and stained with silver. Photographs were taken on electrophoresis duplicating paper; exposure time for lane 1 was shorter than for the others. Numbers at right indicate the position and size (in kDa) of marker proteins that were electrophoresed in an adjacent lane. Lane 1, load of ssDNA cellulose (50 µg); lane 2, eluate of ssDNA cellulose (2.7 µg); lane 3, dRP-A, Mono Q fraction (0.5 µg); lane 4, Dm mtSSB, Mono Q flow-through (0.3 µg); and lane 5, Dm mtSSB, Mono S flow-through (0.3 µg).
That the 18-kDa polypeptide is responsible for DNA binding activity was demonstrated by sedimentation analysis on a glycerol gradient (Fig. 2). When the fractions displaying ssDNA binding were analyzed by silver staining after SDS-polyacrylamide gel electrophoresis, the 18-kDa polypeptide was found to cosediment with the activity (Fig. 2, upper and middle panels). In addition, if these fractions were cross-linked to ssDNA by UV irradiation, a single radiolabeled band was observed upon autoradiography (Fig. 2, lower panel). The radiolabeled band has an apparent molecular mass of 23 kDa, which is slightly larger than the protein itself, a condition which is most likely caused by the addition of oligonucleotides to the polypeptide upon cross-linking. Similar size shifts were observed for other DNA binding proteins, including the large subunit of dRP-A (14) and the tomato golden mosaic virus protein AL1(21) .
Figure 2:
Dm mtSSB consists of a single
polypeptide of 18 kDa. Dm mtSSB (1 µg) was sedimented in a
15-35% glycerol gradient as described under
``Methods.'' Fractions were analyzed for DNA binding by gel
mobility shift assay (upper panel) and UV cross-linking to
radiolabeled ssDNA (lower panel). The protein content of
individual fractions was analyzed by 15% SDS-polyacrylamide gel
electrophoresis and silver staining (middle panel). The band
at 50 kDa that appears in all of the lanes is an artifact of the
silver-staining method. Marker proteins were run in a parallel
gradient, and their positions of migration indicated above the upper
panel.
Figure 3: Dm mtSSB preferentially binds to long DNA. Labeled 32-mer oligonucleotide was mixed with increasing amounts of unlabeled competitor DNAs of various lengths. After addition of Dm mtSSB, gel mobility shift assays were performed as described under ``Methods.'' 17-mer, closed squares; 25-mer, open circles; 32-mer, open squares; 59-mer, closed circles.
Further evaluation of the binding site size was
performed by fluorescence quenching. Analysis of emission spectra
showed that excitation of Dm mtSSB at 284 nm caused maximum
emission at 348 nm (data not shown). Fluorescence is quenched up to 60%
upon addition of ssDNA, indicating interaction of the protein with DNA.
The binding site size was determined from fluorescence titration curves
in which increasing amounts of oligo- or polynucleotides were added
sequentially to fixed amounts of protein. Using Dm mtSSB at
concentrations of 1-5 10
M,
ssM13DNA or 75 mer oligonucleotide were added stepwise to the solution.
Under low salt conditions complex formation was rapid, and equilibrium
was attained within the mixing time. Because under high salt conditions
the rate of binding was slower, readings were taken when the
fluorescence had stabilized after addition of the DNA. Linear
approximations were fitted to the initial and final slopes of the
curves and the intersection of the two lines taken as the apparent
binding site size. Under low salt conditions (50 mM) the
calculated binding site size was 17 ± 3 nt (Fig. 4A). This size did not change significantly at
lower protein concentrations or with different DNAs. However, under
higher salt conditions a significant increase in the binding site size
was observed. Binding site sizes of 28 ± 2 nt and 34 ± 2
nt were determined at 320 mM and 480 mM NaCl,
respectively (Fig. 4A).
Figure 4: Fluorescence titration curves of Dm mtSSB binding to M13 DNA. A, increasing amounts of M13mp7 DNA were added to Dm mtSSB at increasing salt concentrations as described under ``Methods.'' 50 mM NaCl, closed circles; 320 mM NaCl, open circles; 480 mM NaCl, closed triangles. B, after saturation of quenching had been reached, protein-DNA complexes were disrupted by addition of increasing amounts of NaCl. The readings obtained were corrected for the dilution due to addition of the salt solution, and were taken in triplicate.
The salt stability of DNA binding under these conditions was monitored by increasing the NaCl concentration once maximum quenching had been achieved (Fig. 4B). There was no reduction in the quenching observed up to 500 mM NaCl. At 800 mM NaCl, 50% of the initial complex was disrupted, and only 20% of the initial quenching was retained above 1 M NaCl. The residual quenching most likely is due to some tight form of DNA-protein complex, which is not disrupted at this salt concentration.
The same assay was used to examine the ability of Dm mtSSB to bind to dsDNA and RNA (Table 1). While heat-denatured plasmid DNA competed efficiently for binding of the oligonucleotide, the same DNA in its double-stranded form did not. Similarly, tRNA did not compete, suggesting that Dm mtSSB does not bind RNA.
Figure 5:
Dm mtSSB stimulates the rate of
DNA synthesis by Drosophila pol over a broad KCl range. A, DNA synthesis was measured on singly primed M13 DNA as
described under ``Methods,'' in the presence of the indicated
amounts of KCl and in the absence (closed circles) or presence (open circles) of Dm mtSSB (0.8 µg). B,
the data from A were replotted to show the ratio of nucleotide
incorporation by Dm pol
in the presence versus absence of Dm mtSSB at each KCl
concentration.
Figure 6:
Dm mtSSB increases the
processivity of Drosophila pol . DNA synthesis was
performed on singly primed M13 DNA (6407 nucleotides) as described
under ``Methods,'' and the DNA product strands were isolated,
denatured, and electrophoresed on a 1.5% denaturing agarose
gel(16) . Reactions were performed in the absence (lanes
1-3) or presence (lanes 4-6) of Dm mtSSB (2 µg) and incubated at 30 °C for 8 min. Lanes 1 and 4, 30 mM KCl; lanes 2 and 5, 65 mM KCl; lanes 3 and 6, 120
mM KCl. Numbers at left indicate the positions and
sizes (in nt) of HindIII restriction fragments of
-DNA
and HpaII fragments of M13Gori1 DNA (28) that were electrophoresed in an adjacent lane. The
reaction products were also electrophoresed in a 6% denaturing
polyacrylamide gel as described by Williams et
al.(25) , in order to quantitate product DNA strands of
150 nt (data not shown).
Figure 7:
Dm mtSSB increases the rate of
initiation of DNA synthesis by Drosophila pol . Primer
extension was performed at 30 mM KCl in the absence of dCTP as
described under ``Methods.'' DNA product strands were
isolated, denatured, and electrophoresed on an 18% denaturing
polyacrylamide gel(16) . Reactions were performed in the
absence (lane 2) or presence of Dm mtSSB (2 µg, lane 3) or Eco SSB (2 µg, lane 4). Lane 1 represents a control reaction lacking both pol
and SSB.
We have purified the mitochondrial single-stranded DNA-binding protein from D. melanogaster embryos. The identity of Dm mtSSB was demonstrated by amino-terminal sequencing and MALDI-MS analysis of the single 18-kDa polypeptide, and by its localization to a mitochondrial protein fraction. Recently, a cDNA clone of the Dm mtSSB was isolated by screening of an expression library for DNA-binding proteins(22) . The deduced amino acid sequence exhibits a high degree of similarity with those of mtSSBs from X. laevis(8, 24) , yeast(7) , rat(23) , and human tissues (23) and with that of Eco SSB(26) . The cDNA encodes a protein of 15.6 kDa. The amino-terminal sequence of Dm mtSSB and the deduced amino acid sequence of cDNA are identical apart from a leader sequence of 16 aa present in the latter, and MALDI-MS analysis indicates a mass of 13.8 kDa for the mature protein. An overproduced fusion protein generated from the cDNA clone migrates on SDS-polyacrylamide gels with a similar size as Dm mtSSB, and binds preferentially to ssDNA as compared to dsDNA(22) .
We have found that Dm mtSSB binds to ssDNA very tightly, and that binding is resistant to 750 mM NaCl (Fig. 4B), 4 M urea and 0.25% SDS (data not shown). Dm mtSSB discriminates strongly in favor of ssDNA over dsDNA and RNA. Fluorescence quenching titrations indicate a coverage size of 17 nucleotides of DNA per monomer of protein. This is also consistent with the observed mobility shift of Dm mtSSB when it is photocross-linked to DNA. Given a binding site size of 17 nt, it is perhaps surprising that Dm mtSSB does not appear to bind to a 17-mer oligonucleotide. This cannot be explained by cooperativity of the binding reaction, since under low salt conditions Dm mtSSB shows low cooperativity. Instead, a possible explanation is suggested by the observation that on glycerol gradients Dm mtSSB exhibits a sedimentation coefficient of 4.04 S. Thus, the native protein apparently exists as an oligomer, most likely a tetramer, leading to an effective binding site size of 68 nucleotides per tetramer. Under these circumstances, while cooperativity appears to be low between tetramers, the formation of protein-DNA complexes by the individual protomers may be highly cooperative, such that stable binding may not be observed until more than one subunit is bound to the DNA. Consistent with this explanation is the increase in efficiency of binding with an increasing oligonucleotide size of up to at least 59 nt, and a lack of protein: DNA intermediates in gel mobility shift assays that were performed to evaluate binding to the 59 mer (data not shown). We cannot however, rule out entirely the possibility that the methods used for determining the concentration of Dm mtSSB have led to an overestimate of the amount of protein used in the DNA-binding reactions, and therefore an underestimate of binding site size.
The DNA-binding parameters of Dm mtSSB show some variation at higher salt concentrations. An increase in the binding site size from 17 to 28-34 is observed, and there is also an apparent increase in the cooperativity of binding. It therefore seems likely that Dm mtSSB has more than one mode of DNA binding and that the DNA-binding mode is modulated by ionic strength. Several DNA-binding modes resulting in distinct binding site sizes have been demonstrated for Eco SSB(17) . For Eco SSB, changes in cooperativity with varying salt concentrations have also been observed, although in this case binding is with high cooperativity at low salt, while it is low at salt concentrations above 200 mM(18) .
Overall, the physical and biochemical
characteristics of Dm mtSSB are similar to those of Eco SSB (2) and to various features of mtSSBs from rat
liver(5, 9) , X.
laevis(6, 8) , yeast(7) , and a
recombinant form of the human protein(27) . To date, however,
the effects of mtSSB on the function of mitochondrial DNA polymerase
are unclear. The rat (9) and frog (10) mtSSBs have been
shown to stimulate partially purified forms of pol on
homopolymeric DNA substrates, yet the frog protein was found to be
completely inhibitory to mitochondrial DNA polymerase activity on
singly primed single-stranded viral DNA(10) . More recently, an
SSB isolated from human mitochondria was shown to enable highly
purified human pol
to utilize singly primed M13 DNA(11) .
Here, it is curious that the human enzyme alone cannot replicate a
single-stranded DNA template; as a result, the mechanism by which the
SSB exerts its function cannot be readily assessed. At the same time,
it is not known if the human SSB used in that study represents the Eco-like mtSSB reported by others.
We have shown here that
the homogeneous Dm mtSSB stimulates greatly the activity of
its cognate DNA polymerase, the well characterized pol from D. melanogaster embryos(12, 16, 25) . Our primer
extension data show that the stimulation of pol
by Dm mtSSB is due primarily to an increased rate of primer recognition
and binding. Further, Dm mtSSB increases the processivity of Drosophila pol
, reducing or eliminating pausing of DNA
polymerase at specific sites on the template DNA strand where stable
secondary structure is predicted. In fact, the effects of Dm mtSSB on Dm pol
function are strikingly similar to
those found previously for Eco SSB(16) . Taken
together with the high amino acid conservation between mtSSBs and Eco SSB and their similar physical and biochemical properties,
the parallel effects of Dm mtSSB and Eco SSB on mtDNA
polymerase suggest that the important role ascribed to Eco SSB
in bacterial DNA replication also reflects that of mtSSB in the
replication of mitochondrial DNA.