A systematic analysis of mutant Mhr1 proteins revealed that Asp69 is involved in Mg2+-dependent DNA binding, and that multiple Lys and Arg residues located around Trp71 and Trp165 are in
Trang 1mitochondrial ATP-independent homologous pairing
protein
Tokiha Masuda1,2, Feng Ling2, Takehiko Shibata1,2and Tsutomu Mikawa1,2,3
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: mikawa@riken.jp
(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 Mg2+-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.
Trang 2MHR1 is necessary for the homologous
recombina-tion of mtDNA in Saccharomyces cerevisiae The
mhr1-1mutation 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 replicainitia-tion 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
350 300 250
200 150 100 50
0
Wavelength (nm)
0 2 4 6 8 10 12 14 16
A
Fig 1 Fluorescence changes of Mhr1 after
ssDNA binding (A) Schematic
representa-tion of the posirepresenta-tions 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.
Trang 3protein; 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
Wavelength (nm)
250
200
150
100
50
0
160 120 80 40 0
εssDNA (μ M )
At 350 nm
0 2.6 5.2 7.8 10.4 13 15.618.2
At 390 nm
160 120 80 40 0
εssDNA (μ M )
0 2.6 5.2 7.8 10.4 13 15.618.2
0
1 0.8 0.4
0 5 10 15 20 DNA (μ M )
At 350 nm
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 ) at 25 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.
Trang 4fied 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
Wavelength (nm)
400
Wavelength (nm)
400
350 450 500 300 350 400 450 500 300
300
A 9 W t
W71F
W178A W169A
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 l M (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%).
Trang 5the 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
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 0.250.5
0 0.250.5 0 0.250.5 0 0.250.5 0 0.250.5 0 0.250.5
0 0.250.5 0 0.250.5 0 0.250.5
M 0 1 2 M 0 1 2 M 0 1 2 M 0 1 2 M 0 1 2 M 0 1 2
M 0 1 2 M 0 1 2 M 0 1 2
ccc
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).
Trang 6The 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 Mg2+, because the DNA-binding activities of other HP proteins are often increased by the addition of Mg2+ 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
ssDNA
(10 μ M )
well
well
M C
dsDNA
(5 μ M )
K159A/K160A
ssDNA
(10 μ M )
well
well
M C
dsDNA
(5 μ M )
K159A/K160A
0.1 0.2 0.30.40.6
0.1 0.2 0.30.40.6
0.1 0.2 0.3 0.4 0.6
0.1 0.2 0.3 0.4 0.6
0.1 0.2 0.30.40.6
0.1 0.2 0.30.40.6
0.1 0.2 0.3 0.4 0.6
0.1 0.2 0.3 0.4 0.6
0.1 0.2 0.3 0.40.6
0.1 0.2 0.3 0.40.6
0.1 0.2 0.3 0.4 0.6
0.1 0.2 0.3 0.4 0.6
0.1 0.2 0.3 0.4 0.6
0.1 0.2 0.3 0.4 0.6
0.1 0.2 0.3 0.4 0.6
0.1 0.2 0.3 0.4 0.6
0.1 0.2 0.30.4 0.6
0.1 0.2 0.30.4 0.6
dsDNA (10 μ M )
ssDNA (5 μ M )
100
80
60 40 20
0
Mhr1 (μ M )
εssDNA (13 μ M )
well
well
5
5
5
5
E166A D167A
dsDNA (10 μ M )
ssDNA (45 μ M )
M
well
well
0 1 5 10
0 1 5 10
0 1 5 10
0 1 5 10
0 1 5 10 0 1 5 10 0 1 5 10
0 1 5 10
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 l M ) was incubated with the Mhr1 mutants (0.2, 0.4, 0.6, 0.8 and 1.0 l M 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 l M ) was incubated with 0.125, 0.25, 0.5 and 1 l M Mhr1 in T buffer in the presence of 10 m M
EDTA (left) or 5 m M MgCl2(right) at 23 C for 30 min (C) Relative fluorescence changes of essDNA (13 l M ) at 400 nm essDNA (10 l M ) was incubated with varying concentrations of Mhr1 (0, 0.2, 0.4, 0.5, 0.8, 1.6 and 3.0 l M ) in 25 m M Tris ⁄ HCl (pH 7.5) in the presence of
10 m M MgCl2or 10 m M 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 m M 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 m M MgCl 2 ) Circular ssDNA (45 l M , top) or circular dsDNA (10 l M , bottom) was incubated with 0.5 l M (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.
Trang 7included 5 or 10 mm MgCl2 (Fig 5B,C) Therefore,
the DNA-binding activity of Mhr1 was considerably
increased in the presence of MgCl2, although Mg2+
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 Mg2+ (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 MgCl2 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 Mg2+-dependent
DNA-binding activity of Mhr1, as Mg2+increased the
DNA-binding activity of Mhr1 The enhanced DNA-DNA-binding
activity could be due to the shielding of negative
charges around the Mhr1–DNA interface by Mg2+
ions Alternatively, Mhr1 could interact with DNA not
only via its positively charged residues, Lys and Arg, as
discussed above, but also via Mg2+ 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 MgCl2 (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 Mg2+in Fig 5F) These
results suggest that Asp69 is among the residues that are important for the Mg2+-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 (37C) 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 37C, 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
Trang 8Mhr1 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.
Growth on YPGly plates at 37 C
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.
I163Ab
a 25 m M Mes (pH 6.5), 1 m M MgCl2, and 1 m M dithiothreitol b Protein aggregated during the process of purification.
Trang 9finding 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 Mg2+(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
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.
Trang 10Experimental procedures
DNA
A 74-mer ssDNA (5¢-ACGGGTGGGGTGGACATTGAC
GAAGGCTTGGAAGACTTTCCGCCGGAGGAGGAGT
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
Expression and purification of Mhr1 For the expression of recombinant Mhr1, E coli BL21(DE3) pLysS DrecA cells were transformed with the expression
and recombinant protein expression was induced by the
16 h Cells were harvested and suspended in an isotonic
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
1
6 2 1
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 (http://align.bmr.kyushu-u.ac.jp/mafft/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.