Báo cáo khoa học: Analysis of DNA-binding sites on Mhr1, a yeast mitochondrial ATP-independent homologous pairing protein potx

Analysis of DNA-binding sites on Mhr1, a yeast
mitochondrial ATP-independent homologous pairing
protein
Tokiha Masuda
1,2
, Feng Ling
2
, Takehiko Shibata
1,2
and Tsutomu Mikawa
1,2,3
1 Graduate School of Nanobioscience, Yokohama City University, Japan
2 RIKEN Advanced Science Institute, Saitama, Japan
3 RIKEN SPring-8 Center, Hyogo, Japan
Introduction
Homologous DNA recombination is conserved in all
organisms. In the nucleus, homologous recombination
is involved in the maintenance of genome integrity
during mitosis, and in genetic diversification through
meiosis. In bacteria, homologous recombination
strictly depends on the RecA gene [1–4], whereas in
eukaryotes it depends on the Rad51 [5–8] and Dmc1
[9–12] genes, both of which encode RecA orthologs.
Homologous recombination is initiated via a single-
stranded gap or a double-strand break, which is
processed to produce 3¢-ssDNA tails [13]. Each ssDNA
region invades undamaged homologous dsDNA,
resulting in the formation of homologous joints
between the dsDNA and ssDNA through the pairing
of complementary sequences. This reaction is termed
homologous pairing (HP), and it is followed by a

strand exchange to stabilize the joint [1,14]. The
RecA ⁄ Rad51 family of proteins promotes HP, which is
a key process of homologous recombination, in an
ATP-dependent manner in vitro.
Keywords
fluorescence resonance energy transfer
(FRET); homologous recombination; Mhr1;
mtDNA; RecA
Correspondence
T. Mikawa, RIKEN Advanced Science
Institute, 1-7-29 Suehiro-cho, Tsurumi-ku,
Yokohama 230-0045, Japan
Fax: +81 45 5087364
Tel: +81 45 5087224
E-mail:
(Received 6 October 2009, revised 24
December 2009, accepted 8 January 2010)
doi:10.1111/j.1742-4658.2010.07574.x
The Mhr1 protein is necessary for mtDNA homologous recombination in
Saccharomyces cerevisiae. Homologous pairing (HP) is an essential reaction
during homologous recombination, and is generally catalyzed by the
RecA ⁄ Rad51 family of proteins in an ATP-dependent manner. Mhr1 cata-
lyzes HP through a mechanism similar, at the DNA level, to that of the
RecA ⁄ Rad51 proteins, but without utilizing ATP. However, it has no
sequence homology with the RecA ⁄ Rad51 family proteins or with other
ATP-independent HP proteins, and exhibits different requirements for
DNA topology. We are interested in the structural features of the func-
tional domains of Mhr1. In this study, we employed the native fluorescence
of Mhr1’s Trp residues to examine the energy transfer from the Trp resi-
dues to etheno-modified ssDNA bound to Mhr1. Our results showed that

two of the seven Trp residues (Trp71 and Trp165) are spatially close to the
bound DNA. A systematic analysis of mutant Mhr1 proteins revealed that
Asp69 is involved in Mg
2+
-dependent DNA binding, and that multiple Lys
and Arg residues located around Trp71 and Trp165 are involved in the
DNA-binding activity of Mhr1. In addition, in vivo complementation anal-
yses showed that a region around Trp165 is important for the maintenance
of mtDNA. On the basis of these results, we discuss the function of the
region surrounding Trp165.
Abbreviations
dnRad54, Danio rerio Rad54; FRET, fluorescence resonance energy transfer; HP, homologous pairing; essDNA, etheno-ssDNA.
1440 FEBS Journal 277 (2010) 1440–1452 ª 2010 The Authors Journal compilation ª 2010 FEBS
MHR1 is necessary for the homologous recombina-
tion of mtDNA in Saccharomyces cerevisiae. The
mhr1-1 mutation causes defects in mtDNA duplication,
partitioning to bud, and recovery of homoplasmy, all
of which are attributed to the MHR1-dependent initia-
tion of rolling-circle mtDNA replication. This process
occurs through HP, followed by continuous copying of
the complementary sequence of the circular parental
dsDNA [15,16]. The MHR1 gene product, Mhr1, con-
sists of 226 amino acids, binds to both ssDNA and
dsDNA, and catalyzes HP in an ATP-independent
manner in vitro [15,17,18].
In addition to Mhr1, other proteins that promote
HP in an ATP-independent manner have been identi-
fied. These HP proteins include the human Xrcc3–
Rad51c ⁄ Rad51L2 complex (human Rad51 paralogs
[19]), human Rad52 [20], Escherichia coli phage k

b-protein [21], E. coli RecT (a homolog of kb-protein
[22]), E. coli RecO [23], and Ustilago maydis Brh2 [24].
The amino acid sequences and tertiary and quaternary
structures of these ATP-independent HP proteins are
different from those of the RecA ⁄ Rad51 family pro-
teins, and no sequence homologies have been found
among them. HP catalyzed by RecA ⁄ Rad51 is accom-
panied by the untwisting of the dsDNA substrate, and
is strongly stimulated by negative supercoils of the
dsDNA. In contrast, HP by Mhr1 is performed with-
out a net change in the number of dsDNA twists and
is prevented by negative supercoils [25]. However, we
have recently found that RecA ⁄ Rad51 and Mhr1 cause
similar structural changes in the ssDNA, which sug-
gests that they may operate via a common mechanism
at the DNA level [26]. In order to understand the
mechanisms of HP, it will be crucial to determine how
each HP protein causes a similar structural change in
the DNA. These investigations should include the
identification and characterization of the DNA-binding
regions and the binding modes of each of these mole-
cules.
In this study, we analyzed the Mhr1 sites involved
in ssDNA binding, using fluorescence analysis and
site-directed mutagenesis. In a detailed homology
search, we also found that Mhr1 shows partial
sequence similarity to the core helicase domain of
Rad54. Finally, we discuss the DNA-binding mode of
Mhr1 and a potential mechanism for HP.
Results

Quenching of Trp fluorescence after DNA binding
Mhr1 has seven Trp residues (Fig. 1A) and 11 Tyr
residues. Therefore, we examined the binding of Mhr1
to DNA by measuring changes in the fluorescence
spectra of Mhr1 after binding. To distinguish the
fluorescence of Trp from that of Tyr, we selected an
excitation wavelength of 295 nm, because the absorp-
tion associated with Tyr is negligible at this wave-
length. This allowed for the selective examination of
fluorescence from Trp residues [27]. The fluorescence
emission spectra of Mhr1 in the presence and absence
of ssDNA exhibited peaks around 350 nm (Fig. 1B).
The emission spectrum of Mhr1 was quenched when a
74-mer ssDNA was added. Ultimately, the fluorescence
intensity decreased to  60% of the initial intensity
(Fig. 1B). The fluorescence change was saturated at an
ssDNA concentration that was 16-fold greater than
the Mhr1 concentration (8 lm nucleotide⁄ 0.5 lm
160
140
120
100
80
60
40
20
0
ssDNA (µM)
Fluorescence change at 350 nm
305 350 400 450 500

350
300
250
200
150
100
50
0
Wavelength (nm)
Fluorescence intensity
0246810121416
Residue No.
15 59 71 120 165 169 178
A
B
C
Fig. 1. Fluorescence changes of Mhr1 after
ssDNA binding. (A) Schematic representa-
tion of the positions of the Trp residues in
Mhr1. (B) Emission spectra of wild-type
Mhr1 (0.5 l
M) with varying concentrations
of the 74-mer oligo-ssDNA (light gray to
black: 0, 2, 4, 6, 8 and 15 l
M). (C) Fluores-
cence changes of Mhr1 at 350 nm after
ssDNA binding. Measurements were per-
formed three times.
T. Masuda et al. DNA-binding sites of Mhr1
FEBS Journal 277 (2010) 1440–1452 ª 2010 The Authors Journal compilation ª 2010 FEBS 1441

protein; Fig. 1C). These results imply that the environ-
ment of some of the Trp residues changed after the
binding of Mhr1 to the 74-mer ssDNA. This fluores-
cence quenching was also observed in the presence of
shorter ssDNA (a 50-mer and a 34-mer), whereas no
quenching was induced by a 27-mer ssDNA (data not
shown), probably because it was too short for Mhr1
binding. The use of a circular ssDNA molecule as a
substrate (/X174) hampered clear measurement of the
Mhr1 fluorescence spectrum, probably because of scat-
tering from the large Mhr1–ssDNA complex (data not
shown). Although the Trp environment was most
likely affected by the proximity of the DNA, other
possibilities can also be envisioned, such as a confor-
mational change in Mhr1 upon DNA binding. Mhr1
may interact with Mhr1 on the DNA, although it
existed as a monomer in solution (unpublished result).
In this case, the quenching of Trp fluorescence may
occur if some Trp residues are located near the
protein–protein interface.
Fluorescence resonance energy transfer (FRET)
from Mhr1 to etheno-ssDNA (essDNA)
Fluorescence-based assays including FRET analysis
have been applied to the investigation of the nucleo-
tide-binding sites of E. coli RecA [28], T4 phage GP32
[29], and human Rad51 [30]. To examine whether any
Trp residues of Mhr1 are close to the DNA molecule
in the Mhr1–ssDNA complex, we measured the energy
transfer from the Trp residues to the fluorescent nucle-
obase ethenoadenine, which is a fluorescent analog of

the adenine nucleotide. The emission spectrum of Trp
overlaps partially with the absorption spectrum of
essDNA. Therefore, FRET could be used to evaluate
whether a Trp residue was close to the essDNA. The
seven Trp residues of Mhr1 are distributed almost
evenly throughout the polypeptide chain (Fig. 1A).
Thus, a FRET analysis of Mhr1 variants with muta-
tions at the Trp sites should provide information about
the ssDNA-binding region of Mhr1. After the addition
of various amounts of essDNA, we observed signifi-
cant quenching of Trp fluorescence at 350 nm and a
new peak at 390 nm (Fig. 2A), which were considered
to be caused by FRET from Trp to essDNA. As Trp
fluorescence was quenched upon DNA binding
(Fig. 1B), the emission spectra must have comprised
the fluorescence from both quenching and energy
transfer. Therefore, energy transfer from Mhr1 to
essDNA was examined as described previously [29].
When the fluorescence changes at 350 nm in the
Mhr1–essDNA complex were compared with those in
the Mhr1-unmodified DNA complex, essDNA
quenched over 60% of Trp fluorescence, whereas
unmodified DNA quenched < 40% (Fig. 2A, inset).
The addition of essDNA caused over a 1.5-fold
decrease in fluorescence intensity as compared with
unmodified DNA. Thus, FRET from Trp residues to
essDNA was confirmed (Fig. 2A, inset). The changes
in fluorescence intensity (DI) at 350 nm and 390 nm
after essDNA binding were plotted against the concen-
tration of essDNA (Fig. 2B,C). Again, the changes in

fluorescence at 350 and 390 nm were saturated at a
DNA ⁄ Mhr1 concentration ratio of approximately
16 : 1 (7.8 lm nucleotide ⁄ 0.5 lm protein), which was
equal to the saturation ratio obtained using unmodi-
305 350 400 450 500
Wavelength (nm)
Fluorescence intensity
250
200
150
100
50
0
160
120
80
40
0
ΔI
εssDNA (μM)
At 350 nm
0 2.6 5.2 7.8 10.4 13 15.618.2
At 390 nm
160
120
80
40
0
ΔI
εssDNA (μM)

0 2.6 5.2 7.8 10.4 13 15.6 18.2
0
1
0.8
0.4
0 5 10 15 20
DNA (μ
M)
Relative intensity
At 350 nm
A
B
C
Fig. 2. Energy transfer from the Trp resi-
dues of Mhr1 to essDNA. (A) Mhr1 (0.5 l
M)
was incubated with varying concentrations
of essDNA (light gray to black: 0, 2.6, 5.2,
7.8, 10.4, 13, 15.6 and 18.2 l
M)at25°C for
10 min. These samples were excited at
295 nm. The inset shows the relative
fluorescence changes at 350 nm against the
DNA concentrations of the Mhr1–essDNA
complex (filled circle) and Mhr1-unmodified
DNA complex (open circle). Changes in
fluorescence intensity at 350 nm (B) and
390 nm (C) were plotted against essDNA
concentration. Measurements were
performed three times.

DNA-binding sites of Mhr1 T. Masuda et al.
1442 FEBS Journal 277 (2010) 1440–1452 ª 2010 The Authors Journal compilation ª 2010 FEBS
fied ssDNA (Fig. 1B). This result suggested that the
fluorescence modification of ssDNA employed here did
not affect the DNA-binding activity of Mhr1.
FRET of Mhr1 mutants
To identify the Trp residues of Mhr1 that contribute
to the observed FRET, each of the seven Trp residues
was replaced by an Ala, a general candidate for site-
directed mutagenesis. Additionally, Ala is uncharged,
and does not absorb light at 295 nm. Two of these
mutants (W15A and W71A) precipitated during the
purification process, so those Trp residues were
replaced by Phe, which is structurally similar to Trp
but negligibly excited at 295 nm. The seven Mhr1
mutants (W15F, W59A, W71F, W120A, W165A,
W169A, and W178A) were produced in E. coli and
purified using a method similar to the one used to
purify wild-type Mhr1. Figure 3 shows the relative flu-
orescence emission spectra of the seven Mhr1 mutants
in the presence of essDNA. Although all mutants
exhibited energy transfers, the decrease in fluorescence
intensity at 350 nm was much smaller for the W71F
and W165A mutants than it was for the wild type and
other mutants (Fig. 3, gray vertical broken line). This
indicated that the W71F and W165A mutants trans-
ferred energy with less efficiency than the wild type,
and that Trp71 and Trp165 together contributed
significantly to the energy transfer in the wild type.
The results strongly suggested that the DNA-binding

site of Mhr1 occurs near Trp71 and ⁄ or Trp165.
As the fluorescence intensity at 390 nm (Fig. 3, gray
vertical solid line) increased after essDNA addition,
even with the W71F and W165A mutants, the ability
of these mutants to bind ssDNA was expected to be
similar to that observed for the other mutants. To con-
firm this hypothesis, the DNA-binding activity of each
mutants was examined at various protein concentra-
tions. In the absence of Mhr1, no band shift was
detected (arrowheads in Fig. 4). In the presence of
0.5 lm Mhr1 for ssDNA and 1 lm Mhr1 for dsDNA,
the Mhr1–DNA complexes exhibited a complete gel
mobility supershift (arrows in Fig. 4). In the presence
of 0.25 lm Mhr1, about half of the ssDNA complexes
exhibited the supershift (arrows in Fig. 4). For both
ssDNA and dsDNA, the protein ⁄ DNA molecular
ratios required to obtain about a half-shift were
approximately 1 : 40 (Figs S1 and S2). Finally, all of
the mutants showed complete shifts at concentrations
of 0.5 lm (ssDNA) and 1 lm (dsDNA). No mutant
showed weaker DNA-binding activity than the wild
type, although there were slight differences in their
binding activities. These results suggest that no Trp res-
idue was directly involved in DNA binding, and that
80
60
40
20
0
100

80
60
40
20
0
100
80
60
40
20
0
100
Wavelen
g
th (nm)
400350 450
500
Wavelength (nm)
400350 450 500 400350 450
500
300300
300
Relative fluorescence intensity (%)
A95Wtw W15F
W120A
W165A
W71F
W178AW169A
Fig. 3. Energy transfer from the Trp resi-
dues of Mhr1 mutants to essDNA. The

emission spectra of Mhr1 (1 l
M) mutants
were measured in the presence of 0 l
M
(solid line), 5.2 lM (dotted line; this concen-
tration causes roughly a 50% change in
each fluorescence spectrum) and 10.4 l
M
(broken line) essDNA, and were plotted as
relative intensities that were defined as
percentages of the intensity of the Mhr1
mutants alone (i.e. 100%).
T. Masuda et al. DNA-binding sites of Mhr1
FEBS Journal 277 (2010) 1440–1452 ª 2010 The Authors Journal compilation ª 2010 FEBS 1443
the reduction in the efficiency of FRET in the presence
of the W71F or W165A mutants was not due to defects
in the DNA-binding activities of these mutants.
DNA-binding activities of Mhr1 mutants
To examine the effects on DNA-binding activity of
single amino acid substitutions around Trp71 and
Trp165 of Mhr1, the following mutants were prepared
and their DNA-binding activities were measured:
L66A, R67A, R68A, D69A, I70A, K72A, C73A,
S162A, I163A, Y164A, E166A, D167A, P168A,
R170A, and G172A. The I163A mutant became aggre-
gated during the purification process and was not
studied further. All mutants exhibited DNA-binding
activities that were comparable to that of the wild type
in standard buffer conditions (FMG1 buffer) (Figs S1
and S2). These results indicated that it is difficult to

obtain DNA-binding-defective mutants using single-
site mutations. There are many basic amino acids in
these two regions (Arg62, Lys63, Arg67, Arg68, Lys72,
Lys159, Lys160, and Arg170), and multiple residues
may interact with the DNA substrates. Therefore, we
prepared Mhr1 mutants that each contained two or
more substitutions of basic residues around Trp71 or
Trp165, and examined their DNA-binding activities.
wt W59A W120A W165A W169A W178A
MMMMMM
MM
W15F W71F
wt
M
ssDNA
(10 μ
M)
ccc
Well
oc
Well
Lane No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Well
Well
Lane No. 19 20 21 22 23 24 25 26 27
dsDNA
(7 μ
M)
ssDNA
(10 μ

M)
dsDNA
(7 μ
M)
0
0.
25
0
.
5
0
0.
25
0
.
5
0
0.
25
0
.
5
0
0.
25
0
.
5
0
0.

25
0
.
5
0
0.
25
0
.
5
0
0.
25
0
.
5
0
0.
25
0
.
5
0
0
.
25
0
.
5
[Mhr1], μ

μ
M
[Mhr1], μ
μ
M
with 1 mM MgCl
2
MMMMMM
01 2 210210210210210
MMM
01 2 01 2 012
ccc
[Mhr1], μ
μ
M
[Mhr1], μ
μ
M
Fig. 4. DNA-binding activity of the Mhr1 Trp
to Ala ⁄ Phe variants. Circular ssDNA of /
X174 (10 l
M) or circular dsDNA of pUC18
(7 l
M) in FMG1 buffer was incubated with
Mhr1 mutants (0, 0.25 and 0.5 l
M for
ssDNA; 0, 1 and 2 l
M for dsDNA) at 25 °C
for 10 min. The arrowheads indicate the
position of the original DNA band. The

arrows indicate supershifted Mhr1–DNA
complexes (nucleoprotein) stacked in the
wells. oc, open circular dsDNA; ccc, cova-
lently closed circular dsDNA; M, molecular
mass marker (k HindIII).
DNA-binding sites of Mhr1 T. Masuda et al.
1444 FEBS Journal 277 (2010) 1440–1452 ª 2010 The Authors Journal compilation ª 2010 FEBS
The relative DNA-binding activities were assessed by
comparing the amounts of DNA remaining at the
original positions (ssDNA and dsDNA are indicated
by filled and open arrowheads in Fig. 5) and the
amounts of DNA showing intermediate shifts (signals
between the arrowheads and arrows in Fig. 5). Among
the double-site and triple-site mutants prepared, the
R67A ⁄ R68A ⁄ K72A and K159A ⁄ K160A mutants
exhibited clear defects in DNA binding (Fig. 5A),
although their DNA-binding activities were not
completely lost.
We also examined the DNA-binding activity of
Mhr1 in the absence of Mg
2+
, because the DNA-
binding activities of other HP proteins are often
increased by the addition of Mg
2+
in vitro [31,32]. In
the presence of 10 mm EDTA, which is a metal ion-
chelating agent, higher concentrations of Mhr1 were
required for DNA binding than in conditions that
K72A

M C
dsDNA
(5 μ
M)
ssDNA
(10
μ
M)
well
R67A/ R68A
R62A/ K63A
well
wt R67A/R68A/K72A
ssDNA
(10 μ
M)
well
well
M C
dsDNA
(5 μ
M)
K159A/K160A
[Mhr1],
μ
M
0
.
2
0

.
4
0
.
6
0
.
8
1
.
0
0
.
2
0
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4
0
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6
0
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8
1
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0
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2
0

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4
0
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6
0
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8
1
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0
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2
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0
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6
0
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8
1
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0
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2
0

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4
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0
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8
1
.
0
0
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2
0
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4
0
.
6
0
.
8
1
.
0
wt R67A/R68A/K72A
ssDNA
(10 μ
M)

well
well
M C
dsDNA
(5
μ
M)
K159A/K160A
0
.
2
0
.
4
0
.
6
0
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8
1
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0
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2
0
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4
0

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1
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1
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4
0

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6
0
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8
1
.
0
with 1 mM MgCl
2
with 10 mM EDTA
with 10 m
M EDTA
[Mhr1],
μ
M
[Mhr1],
μ
M
[Mhr1],
μ
M
[Mhr1],
μ
M
0
.
2
0
.

4
0
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6
0
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8
1
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1
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1
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1
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0
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0
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1
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2
0
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0
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0
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1
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1
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0

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3
0
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1
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6
5 mM Mg
2+
10 mM EDTA
dsDNA
(10
μ
M)

ssDNA
(5 μ
M)
[Mhr1],
μ
M
100
80
60
40
20
0
Mhr1 (μM)
Relative fluorescence
change (%) at 400 nm
0 0.5 1.0 1.5 2.0 2.5 3.0
10 mM Mg
2+
10 mM EDTA
εssDNA (13 μM)
well
well
0
0
.
1
2
5
0
.

2
5
0
.
5
1
0
0
.
1
2
5
0
.
2
5
0
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5
1
0
0
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1
2
5
0
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2
5

0
.
5
1
0
0
.
1
2
5
0
.
25
0
.
5
1
E166A D167Awt
D69A
[Mg
2+
], mM
dsDNA
(10
μ
M)
ssDNA
(45 μ
M)
M

well
well
01510
01510
01510
01510
01510
01510
01510
01510
Mhr1
(0.5 μ
M)
Mhr1
(0.25 μ
M)
A
B
C
F
D
E
Fig. 5. DNA-binding activity of the Mhr1 Lys ⁄ Arg to Ala variants. (A) Gel mobility shift assay in the presence of Mg
2+
. Circular ssDNA of /
X174 (10 l
M) or circular dsDNA of pUC119 (5 lM) was incubated with the Mhr1 mutants (0.2, 0.4, 0.6, 0.8 and 1.0 lM for ssDNA; 0.1, 0.2,
0.3, 0.4 and 0.6 l
M for dsDNA) in FMG1 buffer at 25 °C for 10 min. (B) Mg
2+

-dependent DNA-binding activity of Mhr1. Circular ssDNA of /
X174 (5 l
M) or circular dsDNA of pUC119 (10 lM) was incubated with 0.125, 0.25, 0.5 and 1 lM Mhr1 in T buffer in the presence of 10 mM
EDTA (left) or 5 mM MgCl
2
(right) at 23 °C for 30 min. (C) Relative fluorescence changes of essDNA (13 lM) at 400 nm. essDNA (10 lM)
was incubated with varying concentrations of Mhr1 (0, 0.2, 0.4, 0.5, 0.8, 1.6 and 3.0 l
M)in25mM Tris ⁄ HCl (pH 7.5) in the presence of
10 m
M MgCl
2
or 10 mM EDTA at 23 °C for 30 min. These samples were excited at 320 nm. Measurements were performed three times.
(D, E) Gel mobility shift assay in the absence of Mg
2+
. The same samples used in (A) were incubated in T buffer with 10 mM EDTA at 23 °C
for 30 min. (F) DNA-binding activity of the Mhr1 Asp ⁄ Glu to Ala variants in the presence of varying concentrations of Mg
2+
[0 (contained
10 m
M EDTA), 1, 5 and 10 mM MgCl
2
). Circular ssDNA (45 lM, top) or circular dsDNA (10 lM, bottom) was incubated with 0.5 lM (for
ssDNA) or 0.25 l
M (for dsDNA) of the Mhr1 mutants in T buffer at 23 °C for 30 min. The arrows indicate supershifted Mhr1–DNA com-
plexes (nucleoprotein) stacked in wells. The arrowheads indicate the original position of DNA. M, molecular mass marker (k HindIII);
C, no-protein control.
T. Masuda et al. DNA-binding sites of Mhr1
FEBS Journal 277 (2010) 1440–1452 ª 2010 The Authors Journal compilation ª 2010 FEBS 1445
included 5 or 10 mm MgCl
2

(Fig. 5B,C). Therefore,
the DNA-binding activity of Mhr1 was considerably
increased in the presence of MgCl
2
, although Mg
2+
mainly affects dsDNA binding. All of the Mhr1
mutants tested exhibited weaker DNA-binding activi-
ties in the presence of 10 mm EDTA than in the pres-
ence of Mg
2+
(compare Fig. 5D,E with Fig. 5A). In
the presence of EDTA, the R67A ⁄ R68A ⁄ K72A and
K159A ⁄ K160A mutants also showed DNA-binding
defects, especially for ssDNA (Fig. 5D). In addition,
we could detect DNA-binding deficiencies in other
Mhr1 mutants that showed no defects in the presence
of MgCl
2
. Whereas the R62A ⁄ K63A mutant was
DNA-binding proficient, the K72A mutant showed
slightly weaker DNA-binding activity than the wild
type, especially for ssDNA, and the R67A ⁄ R68A
mutant showed a clearer defect (Fig. 5E). These
results suggest that the various Lys and Arg residues
form a series of positively charged surfaces at which
Mhr1 interacts with DNA. They also suggested the
presence of multiple DNA-binding sites (at least two
sites near Trp71 and Trp165) on Mhr1. These may
explain our difficulty in obtaining a DNA-binding-

deficient mutant via a single-site mutation.
Next, we focused on the Mg
2+
-dependent DNA-
binding activity of Mhr1, as Mg
2+
increased the DNA-
binding activity of Mhr1. The enhanced DNA-binding
activity could be due to the shielding of negative
charges around the Mhr1–DNA interface by Mg
2+
ions. Alternatively, Mhr1 could interact with DNA not
only via its positively charged residues, Lys and Arg, as
discussed above, but also via Mg
2+
ions. To further
explore the effects of charged amino acids on DNA
binding, we replaced the acidic amino acids surround-
ing Trp71 and Trp165 with Ala, and examined the
binding activities of these mutants. The E166A and
D167A mutants exhibited higher affinities for both
ssDNA and dsDNA than the wild type (Fig. 5F), prob-
ably because the mutations reduced the negative
charge, which generally repels the negatively charged
DNA backbone. However, the D69A mutant, which
also had a decreased negative charge, exhibited weaker
DNA-binding affinity than the wild type (Fig. 5F). At
the highest concentration of MgCl
2
(10 mm), the

dsDNA in the presence of the D69A mutant remained
at its original position, whereas the wild type and the
E166A and D167A mutants produced almost complete
supershifts (open arrowhead in Fig. 5F). The D69A
mutant showed slightly weaker ssDNA-binding affinity
than the wild type (filled arrowhead in Fig. 5F),
whereas the E166A and D167A mutants exhibited
higher ssDNA-binding affinities than the wild type
(lane corresponding to 1 mm Mg
2+
in Fig. 5F). These
results suggest that Asp69 is among the residues that
are important for the Mg
2+
-dependent DNA binding
of Mhr1. The DNA-binding activities of all the Mhr1
variants examined are summarized in Table 1.
In vivo complementation assay
The mhr1-1 yeast mutant is the only functionally
defective (in vivo) MHR1 mutant isolated to date. It
has defects in mtDNA recombination, which is neces-
sary for the maintenance of mtDNA. The mutant
gene product has a single amino acid replacement
(G172D) and exhibits defects in HP in vitro [18]. In
this study, we demonstrated that Trp165, near
Gly172, is close to the DNA in the Mhr1–DNA com-
plex. Therefore, we expected that the region
surrounding Trp165 would also play an important
role in vivo. To test this hypothesis, we used the
Mhr1 mutant proteins that had amino acid replace-

ments in this region in complementation assays with
the mhr1-1 mutant cells (FL67-1423 [18]). Comple-
mentation was evaluated by examining the respiration
defect phenotype (see Experimental procedures). The
mhr1 mutant constructs were overexpressed in the
yeast mhr1-1 cells using pRS416 vectors (Table 2
[17]). The mhr1-1 cells that were transformed with
empty vector failed to grow on glycerol medium
[yeast extract ⁄ peptone ⁄ glycerol (YPGly)] at a nonper-
missive temperature (37 °C). However, mhr1-1 cells
that expressed wild-type MHR1 grew under these
conditions (Fig. 6). The mhr1-1 cells transformed with
the S162A, Y164A and P168A mutants also grew on
YPGly at 37 °C, whereas the cells transformed with
the other mutants did not (Fig. 6; Table 2). These
results suggest that Ile163, Trp165, Glu166, Asp167,
Trp169, Arg170 and Gly172 play a role in mtDNA
maintenance in vivo. This high frequency of important
residues around Trp165 (seven of 10 residues) may be
related to the fact that Trp165 is located near the
DNA-binding site (see Discussion).
A search for proteins with homology to Mhr1
To date, there have been no reports on the three-
dimensional structure of Mhr1 or on any sequence or
structural homology between Mhr1 and other proteins.
Therefore, the functional domains of Mhr1 are difficult
to predict. To acquire structural information on Mhr1,
we performed a detailed homology search. Unexpect-
edly, we found that the central portion of Mhr1 (resi-
dues 77–217) exhibits sequence homology with the

C-terminal RecA-like domain of zebrafish (Danio rerio)
Rad54 (dnRad54) (residues 510–649), suggesting that
DNA-binding sites of Mhr1 T. Masuda et al.
1446 FEBS Journal 277 (2010) 1440–1452 ª 2010 The Authors Journal compilation ª 2010 FEBS
Mhr1 shares a RecA-like domain with dnRad54
(Fig. 7A). The identity and similarity in this region
were 22.0% and 42.6%, respectively (the similarity
matrix used was BLOSUM62).
Discussion
In this study, we demonstrated that two distinct regions
of Mhr1, a region around Trp165 (containing Lys159
and Lys160) and one around Trp71 (containing Arg67,
Arg68, Asp69, and Lys72), are important in DNA
recognition, and that Mhr1 has partial homology with
dnRad54, a conserved protein involved in Rad51-medi-
ated homologous recombination [33,34]. Rad54
contains an SWI2 ⁄ SNF2 chromatin-remodeling domain
that includes two RecA-like helicase domains (Fig. 7B
[35]). The cleft between the two RecA-like helicase
domains is predicted to be a DNA-binding surface
[35,36]. This feature is also found in other superfamily 1
and superfamily 2 helicases (e.g. RecQ, UvrB, RecG,
and PcrA) [37–39]. The first RecA-like domain, which is
positioned at the N-terminus of dnRad54, contains a
putative ATP-binding site (WalkerA motif); however,
the second RecA-like domain, positioned at the C-termi-
nus, does not [35]. Mhr1 exhibits sequence homology
with the second RecA-like domain of dnRad54. This
Table 2. Effect of the mutations surrounding Trp165 on the growth
of mhr1-1 cells.

Vector name Mutation
Growth on YPGly
plates at 37 °C
pRS416CM_162 S162A +
pRS416CM_163 I163A )
pRS416CM_164 Y164A +
pRS416CM_165 W165A )
pRS416CM_166 E166A )
pRS416CM_167 D167A )
pRS416CM_168 P168A +
pRS416CM_169 W169A )
pRS416CM_170 R170A )
pRS416CM_172 G172A )
pRS416CM Wild type +
pRS416 Control vector )
Table 1. DNA-binding activity of the Mhr1 variants. N, DNA-binding activity is comparable to that of the wild type; +, DNA-binding activity is
slightly stronger than that of the wild type; +++, DNA-binding activity is clearly stronger than that of the wild type; ), DNA-binding activity is
slightly weaker than that of the wild type; ))), DNA-binding activity is clearly weaker than that of the wild type; )), DNA-binding activity
is weaker than that of the wild type.
Normal condition
a
)MgCl
2
+MgCl
2
Mutant name ss ds ss ds ss ds Fig. no.
W15F N N 4
W59A N N 4
W71F N N 4
W120A N N 4

W165A N N 4
W169A N N 4
W178A N N 4
L66A + + S1
R67A N ) S1
R68A N N S1
D69A N N ) ))) S1, 5F
I70A N N S1
K72A N N ) N S1, 5E
C73A + + S1
S162A N N S2
I163A
b
Y164A + N S2
E166A + N + + + + + + S2, 5F
D167A N N + + + + + + S2, 5F
P168A N N S2
R170A N N S2
G172A N N S2
R62A ⁄ K63A N N 5E
R67A ⁄ R68A ))) ))) 5E
R67A ⁄ R68A ⁄ K72A ))) ))) ))) )) 5A,D
K159A ⁄ K160A ))) ))) ))) )) 5A,D
a
25 mM Mes (pH 6.5), 1 mM MgCl
2
, and 1 mM dithiothreitol.
b
Protein aggregated during the process of purification.
T. Masuda et al. DNA-binding sites of Mhr1

FEBS Journal 277 (2010) 1440–1452 ª 2010 The Authors Journal compilation ª 2010 FEBS 1447
finding is consistent with the absence of the requirement
for ATP in Mhr1-catalyzed HP [18].
RecA-like domains, some of which have an aromatic-
rich loop, generally consist of several parallel b-sheets,
and a-helices that surround the b-sheets. The aromatic-
rich loop in the first RecA-like domain of RecQ is
important for the linking of ATP hydrolysis to DNA
binding ⁄ unwinding [40]. Amino acid substitutions in the
aromatic-rich loop of RecQ modify its ATP hydrolysis
activity and reduce its DNA-unwinding (helicase) activ-
ity [40]. The crystal structure of the PcrA–DNA com-
plex shows that the residues of the aromatic-rich loop
stack with a base in the ssDNA [41]. In this study, we
found that the region around Trp165 of Mhr1 has an
aromatic-rich sequence (residues 163–167: IYWED), is
close to the DNA, and is important for the maintenance
of mtDNA in vivo (Figs 3, 5 and 6; Table 1). There is no
evidence indicating that the aromatic-rich region of
Mhr1 forms a loop; however, this region may function
in a fashion similar to that of RecQ and ⁄ or PcrA. The
L2 loop of E. coli RecA, a DNA-binding region, has
also been examined by mutagenesis and FRET analysis
[28]. Although F203W, a mutation in the central region
of the L2 loop, would be close to DNA, a large FRET
from Trp203 to poly(deoxy-ethenoadenine) was not
observed, probably because of their unfavorable relative
orientations. This notion is supported by the recent crys-
tal structure of the RecA–ssDNA complex, which shows
that the side chain of Phe203 is oriented vertically

towards the DNA bases, although the bases and side
chain are in close proximity to each other [42]. There-
fore, Trp165 (and also Trp71) of Mhr1 may be oriented
horizontally towards the DNA bases, a condition favor-
able for FRET.
Homology modeling of the Mhr1 core (residues 77–
217), based on the sequence alignment between Mhr1
and dnRad54 (Fig. 7A), predicted that Mhr1 has a
RecA-like helicase domain (Fig. 7C). In the model, the
aromatic-rich region (residues 163–167) forms a loop
that protrudes from the core structure (Fig. 7C;
Fig. S3). Thus, the model structure supports the exis-
tence of an aromatic-rich loop in Mhr1. The proteins
with the mutations E166A and D167A in the aro-
matic-rich region, which led to the loss of negative
charges, exhibited higher affinities for DNA than the
wild type (Fig. 5). In contrast, the protein with the
K159A ⁄ K160A double mutation, which led to the loss
of positive charges, showed weaker affinity for DNA
than the wild type. Therefore, after DNA binding, this
region would form a structure that recognizes the neg-
atively charged sugar–phosphate DNA backbone. The
strong defect of mhr1-1 (G172D) may also be due to
the introduction of a negative charge in this region.
Regarding the region around Trp71, Asp69 seems to
interact with DNA via Mg
2+
(Fig. 5F), whereas Arg67,
Arg68 and Lys72 interact directly with the DNA
(Fig. 5). Therefore, these residues form a positively

charged surface that interacts with the sugar–phosphate
backbone. However, we could not predict the spatial
orientation and the tertiary structure of this region, as
there was no sequence homology between this region
and dnRad54 or any other protein in the database.
On the basis of the results from this study, we pro-
pose that the regions around Trp71 (especially Arg67,
Arg68, Asp69, and Lys72) and Trp165 (especially
Lys159 and Lys160) of Mhr1 interact with DNA, and
that the region around Trp165 (i.e. the putative aro-
matic-rich loop) may undergo a conformational change
that occurs after DNA binding. This conformational
change may be important for Mhr1 function, although
this will have to be confirmed via the elucidation of
the tertiary structure of Mhr1.
SD-Uracil
YPGly
30 °C
YPGly
37 °C
wt
c
162
163 164
165
167
168
169
170
172

178
166
Fig. 6. In vivo complementation experiments in mhr1-1 cells using
the mhr1 mutants with changes surrounding Trp165. All transfor-
mants were grown on synthetic defined (SD) uracil plates at 30 °C.
The colonies on the master plate were replicated on two YPGly
plates. These plates were incubated at 30 °C and 37 °C. c, wt and
each number indicate control vector [FL67-1423 ⁄ pRS416 (URA3)],
wild-type MHR1 [FL67-1423 ⁄ pRS416CM (MHR1, URA3)] and the
amino acid number at each mutation site, respectively. All residues
listed were replaced with an Ala.
DNA-binding sites of Mhr1 T. Masuda et al.
1448 FEBS Journal 277 (2010) 1440–1452 ª 2010 The Authors Journal compilation ª 2010 FEBS
Experimental procedures
DNA
A 74-mer ssDNA (5¢-ACGGGTGGGGTGGACATTGAC
GAAGGCTTGGAAGACTTTCCGCCGGAGGAGGAGT
TGCCGTTTTAATAAGGATC-3¢) (Hokkaido System
Science, Hokkaido, Japan) and /X174 circular ssDNA
(New England BioLabs, MA, USA) were purchased com-
mercially. The essDNA was prepared as described in the
literature [43–45], with minor modifications, and its concen-
tration was determined using an e
260 nm
value of
7.0 · 10
3
m
)1
Æcm

)1
.
Expression and purification of Mhr1
For the expression of recombinant Mhr1, E. coli BL21(DE3)
pLysS DrecA cells were transformed with the expression
vector pET14b–Mhr1 [18]. Cells were grown at 37 °C for 4 h,
and recombinant protein expression was induced by the addi-
tion of 1 mm isopropyl thio-b-d-galactoside at 18 °C for
16 h. Cells were harvested and suspended in an isotonic solu-
tion [25 mm Tris ⁄ HCl (pH 8.0), 50 mm glucose, 5 mm 2-mer-
captoethanol, and 0.1 mm p-amidinophenylmethanesulfonyl
fluoride hydrochloride]. Cells were disrupted by sonication
on ice, and 0.5 m NaCl was then added to the lysate. After
centrifugation (45 min at 60 000 g), the supernatant was
Putative aromatic-rich loop
(163-167: IYWED)
N
RecA-like domain 1 (N-terminal) RecA-like domain 2 (C-terminal)
dnRad54
Mhr1
NTD
c
CTD
HD1
HD2
HD2
77 217
91 619 7371668747934511
6221
Mhr1 (77-217)

Aromatic-rich region
A
B
C
Fig. 7. Sequence alignment of Mhr1 and dnRad54. (A) Sequence alignment between dnRad54 and Mhr1 using MAFFT alignment software
( Black and gray boxes indicate identity and similarity, respectively. (B) Location of individual
domains of dnRad54b and Mhr1. Domains are colored yellow (NTD, N-terminal domain), blue [RecA-like domain (N-terminal)], light blue
(HD1, helical domain 1), pink (HD2, helical domain 2), red [RecA-like domain (C-terminal)], and purple (CTD, C-terminal domain). (C) Model
structure of the Mhr1 core. Residues 510–649 of dnRad54 were used as a reference structure. The putative aromatic-rich loop (resi-
dues 163–167: IYWED) is colored purple.
T. Masuda et al. DNA-binding sites of Mhr1
FEBS Journal 277 (2010) 1440–1452 ª 2010 The Authors Journal compilation ª 2010 FEBS 1449
loaded onto an Ni
2+
–nitrilotriacetic acid Superflow (Qiagen
GmbH, Hilden, Germany) column with NB buffer [50 mm
Na
3
PO
4
(pH 7.0), 500 mm NaCl, 5 mm imidazole, 10% glyc-
erol, and 0.01 mm p-amidinophenylmethanesulfonyl fluoride
hydrochloride]. The column was washed with NW buffer
[50 mm Na
3
PO
4
(pH 7.0), 60 mm imidazole, 10% glycerol,
and 0.01 mm p-amidinophenylmethanesulfonyl fluoride
hydrochloride], and Mhr1 was eluted using a linear imidazole

gradient (60–400 mm). Mhr1 fractions were diluted four-fold
in SPA buffer [25 mm Tris ⁄ HCl (pH 7.5), 75 mm NaCl, 10%
glycerol, 1 mm EDTA, 5 mm 2-mercaptoethanol, and
0.01 mm p-amidinophenylmethanesulfonyl fluoride hydro-
chloride] and loaded onto an SP Sepharose Fastflow (GE
Healthcare, Chalfont St Giles, UK) column pre-equilibrated
with SPA buffer. Mhr1 was eluted using a linear NaCl gradi-
ent (75–1000 mm). Mhr1 fractions were dialyzed against
25 mm Tris ⁄ HCl (pH 7.5), 10% glycerol, and 1 mm EDTA.
After cleavage of the His tag, Mhr1 was concentrated and
then stored at )20 °C prior to use.
Fluorescence measurements
All fluorescence measurements were initiated after incuba-
tion of cuvettes in a luminescence spectrometer (Perkin
Elmer, Waltham, MA, USA; LS 55, equipped with a
temperature-regulated holder) for 1 min. The excitation
wavelength was set at 295 nm for selective excitation of
Trp residues, or at 320 nm for excitation of essDNA.
For analysis of the quenching of Trp fluorescence, Mhr1–
DNA complexes were formed by addition of 74-mer oligo-
DNA to 0.5 lm Mhr1 in FMG1 buffer [25 mm Mes (pH 6.5),
1mm MgCl
2
, and 1 mm dithiothreitol]. The mixture was incu-
bated for 5 min at 25 °C, and then placed in the quartz cuv-
ette. For the analysis of FRET, essDNA was added to 0.5 lm
Mhr1 in FMG1 buffer, and incubated for 5 min at 25 °C. The
fluorescence intensity of essDNA alone at each concentration
was subtracted from the total fluorescence intensity of the
essDNA–Mhr1 complexes. For analysis of Mg

2+
depen-
dency, Mhr1–essDNA complexes were formed by addition of
Mhr1 to 13 lm essDNA in 25 mm Tris ⁄ HCl (pH 7.5) in the
presence of 10 mm MgCl
2
or 10 mm EDTA. The Mhr1–ess
DNA complexes were incubated for 30 min at 23 °C.
Site-directed mutagenesis
Single amino acid mutagenesis was performed using the
QuikChange Mutagenesis kit (Stratagene, CA, USA).
Electrophoretic mobility shift assay
Generally, Mhr1–DNA complexes were formed in FMG1
buffer for 10 min at 25 °C. For Mg
2+
dependency experi-
ments (Fig. 5B–F), DNA substrates were incubated with
Mhr1 for 30 min at 23 °C in T buffer [25 mm Tris ⁄ HCl
(pH 7.5) and 0.01% BSA] in the presence of 1, 5 and 10 mm
MgCl
2
or 10 mm EDTA. The protein–DNA complexes were
resolved by 0.7% agarose gel electrophoresis in TAE buffer
[40 mm Tris ⁄ acetate (pH 8.0) and 1 mm EDTA]. After gel
electrophoresis, the bands were visualized using the GelStar
nucleic acid gel stain (Lonza Group, Switzerland).
In vivo complementation assay
The yeast strain and the media used were as described previ-
ously [15,17,18]. The DNA fragment containing the muta-
tion site of Mhr1 was excised from the pET14b–MHR1

plasmid using BmgBI (New England BioLabs) and HindIII
(Takara Bio, Shiga, Japan). This fragment was inserted into
the pRS416CM (MHR1, URA3) expression vector [18], using
the same restriction sites. The yeast mhr1-1 strain (FL67-
1423Dura3) was transformed with pRS416, pRS416CM, and
the mutant plasmids. S. cerevisiae mhr1-1 cells exhibit defects
in mtDNA recombination and in the maintenance of func-
tional mtDNA at 37 °C. At 37 °C, respiration-deficient cells
grow on yeast extract ⁄ peptone ⁄ dextrose (YPD) plates, but
not on YPGly plates, whereas respiration-proficient cells
grow on both types of plates. All transformants were grown
on a synthetic defined uracil plate at 30 °C. Colonies on the
master plate were replicated onto two YPGly plates. These
plates were incubated at 30 °C and 37 ° C.
Homology modeling
A structural model of Mhr1 was generated using the com-
parative homology modeling software modeller , ver-
sion 8.2 [46,47]. The structure of Rad54 (Protein Data
Bank ID: 1Z3I) was obtained from the Protein Data Bank
and was employed as a basal structure. A molecular dia-
gram was generated using ucsf chimera [48].
Acknowledgements
This work was supported in part by Grants-in-Aid
from the Japan Society for the Promotion of Science
(JSPS) to T. Shibata and T. Mikawa. Funding for the
Open Access publication charges for this article was
also provided by the JSPS.
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Supporting information
The following supplementary materials are available:
Fig. S1. DNA-binding activity of Mhr1 variants around
Trp71.
Fig. S2. DNA-binding activity of Mhr1 variants around
Trp165.
Fig. S3. Positions of Trp residues and acidic residues
on putative loop region in Mhr1 core region model.
This supplementary material can be found in the
online version of this article.
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should be addressed to the authors.
DNA-binding sites of Mhr1 T. Masuda et al.
1452 FEBS Journal 277 (2010) 1440–1452 ª 2010 The Authors Journal compilation ª 2010 FEBS