From the Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany
Received for publication, December 2, 2002
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ABSTRACT |
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The RING finger protein RAD5 interacts and
cooperates with the UBC13-MMS2 ubiquitin-conjugating enzyme in
postreplication DNA damage repair in yeast. Previous observations
implied that the function of UBC13 and MMS2 is dependent on the
presence of RAD5, suggesting that the RING finger protein might act as
a ubiquitin-protein ligase specific for the UBC13-MMS2 complex. In
support of this notion it is shown here that the contact surfaces
between the RAD5 RING domain and UBC13 correspond to those found in
other pairs of ubiquitin-conjugating enzymes and ubiquitin-protein
ligases. Mutations that compromise the protein-protein interactions
either between the RING domain and UBC13 or within the UBC13-MMS2 dimer were found to have variable effects on repair activity in
vivo that strongly depended on the expression levels of the
corresponding mutants. Quantitative analysis of the affinity and
kinetics of the UBC13-MMS2 interaction suggests a highly dynamic
association model in which compromised mutual interactions result in
phenotypic effects only under conditions where protein levels become
limiting. Finally, this study demonstrates that beyond its cooperation
with the UBC13-MMS2 dimer, RAD5 must have an additional role in DNA damage repair independent of its RING finger domain.
Covalent attachment of ubiquitin to a target protein
generates a signal that can function in the regulation of many
biological processes, ranging from cell cycle progression and
transcriptional activation to inflammatory and immune responses (1-4).
Ubiquitylation of a protein is a multistep reaction involving a complex
enzymatic cascade (5); in an ATP-dependent reaction, the C
terminus of ubiquitin is linked via a thioester bond to a cysteine
residue in the active site of ubiquitin-activating enzyme
(E1).1 The ubiquitin
thioester is then transferred to the active site cysteine of an E2,
which mediates the attachment of the ubiquitin C terminus to a lysine
residue of the target protein, resulting in an isopeptide bond. This
last step is most often catalyzed by another enzyme, termed
ubiquitin-protein ligase or E3. Repeated rounds of ubiquitylation
result in the formation of long multiubiquitin chains in which each
ubiquitin moiety is linked to an internal lysine residue, usually
Lys48, of the preceding one (6). All of the
eukaryotic genomes encode multiple E2s and an even larger number of
E3s, the latter being responsible for substrate recognition (5, 7, 8).
One prominent class of ubiquitin protein ligases is characterized by
the presence of a RING domain, a specialized type of zinc finger in
which two Zn2+ ions are coordinated by a group of cysteine
or histidine residues in a characteristic arrangement (9-11). The RING
finger has been proposed to fulfill a scaffold function and has in many
cases been shown to be involved in E2 binding (12-14). The x-ray
structure of the mammalian E3 c-Cbl in complex with a cognate E2,
UbcH7, has given insight into the molecular details of the RING
domain-E2 interaction (15). A similar contact surface was found by NMR analysis in another RING finger E3, CNOT4 (16, 17). Hundreds of RING
finger proteins have been described to date; however, although a
growing number of them is being identified as ubiquitin ligases (12,
13, 16, 18), by no means all of them have been shown to possess E3
activity or be involved in ubiquitylation at all, raising the question
of what characteristics distinguish ubiquitin ligases from other RING
finger proteins.
Modification by ubiquitin generally marks a protein for degradation by
the 26 S proteasome (3). However, ubiquitylation can also convey
nonproteolytic signals (2, 19-21). In particular, nonstandard
multiubiquitin chains in which one ubiquitin moiety is linked to the
next via Lys63 have been implicated in such diverse
processes as DNA damage repair (22), endocytosis (23), ribosome
biogenesis (24), mitochondrial inheritance (25), and NF Yeast Strains and Media--
The wt yeast strain used
in this study as well as the isogenic mutants
ubc13::HIS3,
rad5::HIS3, and
ubc13::HIS3
rad5::HIS3 have been described previously
(36). Strain PJ69-4A (38) was used for two-hybrid assays. Standard
protocols were followed for the preparation of yeast media and
transformations (39). SC medium was a modification from that described
by Guthrie and Fink (39) and contained 100 mg/liter of each amino acid
except for leucine, which was present at 200 mg/liter. This corresponds
to a cysteine concentration of 0.67 mM. Yeast strains
harboring integrative plasmids were propagated in YPD medium following
the initial selection on solid and in liquid medium after
transformation; strains with centromeric plasmids were maintained in
selective SC medium at all times.
Construction of Plasmids--
Two-hybrid constructs bearing the
ORFs of RAD5, UBC13, and MMS2 have
been described (36). Site-directed mutants were generated by polymerase
chain reaction and recloned into the two-hybrid vectors, and the
amplified regions were fully sequenced. The intron was removed during
construction of the UBC13 mutants; however, its presence or
absence did not affect protein
levels.2 For expression under
the control of the native promoter, the RAD5 ORF and the
RING finger mutants were recloned from the two-hybrid vectors as
BamHI/PstI fragments into a derivative of the
integrative vector YIplac211 (40) carrying an
EcoRI/BamHI fragment encompassing 245 bp of the
RAD5 upstream region and a PstI/SphI
fragment derived from pGBT9 (Clontech) as a
transcriptional terminator (YIp211-PRAD5-RAD5). UBC13 and its mutants were expressed under the control of
the native promoter in a similar vector in which 996 bp of the
UBC13 upstream regions were inserted as an
EcoRI/BamHI fragment upstream of the ORF
(YIp211-PUBC13-UBC13). Regulatable expression levels were
achieved by placing the UBC13 wt or mutant ORFs under the control of the MET3 promoter into the centromeric vector
YCplac111 (40), again in combination with the pGBT9-derived
transcriptional terminator (YCp111-PMET3-UBC13).
For the production of recombinant proteins in Escherichia
coli, UBC13 and MMS2 as well as the
respective mutants were recloned into expression vectors, allowing the
production of different fusion proteins. pGEX-4T-1 (Amersham
Biosciences) was used to produce N-terminal GST fusions of UBC13 and
its mutants, MMS2, and GST alone. pQE-30 (Qiagen) served as an
expression vector for His6-tagged UBC13. The MMS2 ORF as
well as the mutant F8A were inserted into the vector pTYB12 (New
England Biolabs). This vector affords expression of MMS2 as an
N-terminal fusion to a chitin-binding domain, linked by the
self-cleavable VMA1 intein sequence, which allows
single-step purification of MMS2 bearing three additional amino acids
(AGH) at its N terminus. For simplicity, this construct will be
referred to as MMS2 in the following text. Sequence maps of all the
constructs used in this study are available on request.
Two-hybrid Assays--
Analysis of the interactions between
RAD5, UBC13, and MMS2 in the two-hybrid system was performed as
described previously, using growth on histidine-selective medium as an
indication for a positive interaction (36).
Determination of UV Sensitivities--
UV sensitivities were
determined by means of survival curves or gradient plate assays as
described previously (36, 41). All of the irradiations were performed
at 1.67 J m Analysis of Protein Levels in Yeast--
Yeast cultures were
grown at 28 °C to exponential phase. Samples of 108
cells were lysed by NaOH/ Preparation of Proteins and Antibodies--
Recombinant proteins
were produced in E. coli BL21(DE3) harboring the respective
expression constructs by induction with 1 mM
isopropyl- Protein Interaction Experiments--
Interaction between UBC13
and MMS2 was analyzed by surface plasmon resonance using a Biacore X
instrument. The proteins were dialyzed against HBS buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM
EDTA, 0.05% P-20 detergent). A CM5 chip was derivatized with 15,000 RU
of an anti-GST antibody (Biacore) by amine coupling according to the
manufacturer's recommendation. Subsequently, ~850 RU of the GST
fusion protein (ligand) were immobilized in one of the flow cells. A
reference surface was generated by loading of ~500 RU of
underivatized GST in the other flow cell. The signal in this reference
cell was subtracted online during all measurements. The soluble binding
partner (analyte) was injected at a range of concentrations in 2-min
pulses at a flow rate of 5 µl/min. Because of the fast dissociation
rates, no regeneration of the chip surface was necessary between
individual injections. After analysis of one particular set of
proteins, the ligand was removed from the surface by a 2-min pulse of
10 mM glycine, pH 2.2, followed by immobilization of the
next GST fusion protein to be analyzed. The signal at equilibrium
(RUeq) was plotted against the analyte concentration C, and the steady state affinity KA
and dissociation constant KD were determined by a
fit of this plot according to the model RUeq = KA·C·RUmax/(1 + KA·C), where
RUmax is the theoretical binding capacity at
infinite analyte concentration and KD = 1/KA. Following the BiaEvaluationTM
program, the fits were considered acceptable if the Ubiquitin Conjugation Assays--
Ubiquitin chain synthesis was
assayed at 30 °C in 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 10 mM ATP, and 10 mM dithiothreitol. Yeast E1
(Affiniti Research) was used at a concentration of 70 nM.
Bovine ubiquitin (Sigma) was present at 0.5 mg/ml (58 µM). The reactions were set up with equimolar amounts of
GST-UBC13 and MMS2 (0.5 or 5.0 µM each). The samples were
withdrawn after 0, 1, 2, and 4 h, and the aliquots were analyzed
by SDS-PAGE and Western blots, using an anti-ubiquitin antiserum (Sigma).
Design of Mutations in the Putative Contact Surfaces of RAD5 and
UBC13--
Site-directed mutagenesis of UBC13 and the RAD5 RING domain
was guided by structural information on the UBC13-MMS2 dimer (43) as
well as two mammalian RING finger ubiquitin-protein ligases in complex
with their cognate E2s, c-Cbl with UbcH7 (15) and CNOT4 with UbcH5
(16). Sequence alignment of the RAD5 RING finger with the corresponding
domains of c-Cbl and CNOT4 (Fig.
1A) revealed sufficient
homology to allow the design of RAD5 mutations predicted to influence
the contact with UBC13. Mutation C914S, which affects one of the
cysteines involved in Zn2+ coordination, had previously
been shown to disrupt interaction with the E2 (36). Because this change
is likely to disturb the integrity of the entire domain, additional
more subtle mutations were designed by changing predicted surface
residues to alanine. Residue Ile916 is conserved in many
RING domains, and the corresponding amino acids in both c-Cbl and CNOT4
contact the respective E2. Positions Glu943 and
Tyr944 could be aligned with the interface residues
Ser407 and Trp408 in c-Cbl, and
Asn959 again corresponds to a position involved in E2
contacts in both c-Cbl and CNOT4. In addition, D923A was designed as a
control mutation representing a hydrophilic, thus likely
surface-exposed residue distant to the putative E2 interface.
In the c-Cbl-UbcH7 complex the E2 contacts the RING finger and adjacent
regions by means of charged amino acids within its N-terminal Point Mutations in RAD5 and UBC13 Abolish Association of the Two
Proteins--
The mutants designed within the RAD5 RING finger were
analyzed with respect to their interactions with UBC13 in the
two-hybrid system. This method has been used previously to demonstrate
the interaction between the two proteins and correlates well with results obtained by co-immunoprecipitation (36). To exclude the
possibility that a negative result might be due to improper folding or
expression of the mutant protein in the two-hybrid system, dimerization
with RAD5 itself, which is independent of the RAD5-UBC13 contact, was
assayed in parallel. Fig. 2A
shows that all of the mutants retained association with RAD5 itself; however, they varied considerably in their ability to interact with
UBC13. Mutation C914S, an exchange likely to disrupt the structural
integrity of the entire RING domain, had previously been shown to
prevent the interaction with UBC13 (36). Mutation D923A had no effect
on association with UBC13, which was expected because this residue
should occupy a position in the RING finger facing away from the
putative E2 contact site. Mutations I916A, Y944A, and N959A, all
predicted to reside on the surface of the RING domain facing the E2,
indeed abolished interaction with UBC13 in the two-hybrid system. In
contrast, mutation of Glu943, also predicted to contribute
to the E2 interface, to alanine did not prevent interaction with UBC13.
Thus, the interaction of the RAD5 RING finger with UBC13 involves a
surface similar but not identical to that within other RING domains,
including several highly conserved residues but also showing variations that are likely to affect the respective E2 specificity.
In the reciprocal approach, the ability of the putative contact site
mutants of UBC13 to interact with RAD5 was examined. Interaction with
MMS2, which is independent of the UBC13-RAD5 association (36), was
examined in parallel to ensure proper function of the mutants in the
two-hybrid system. UBC13(E55A) served as a control for a protein that
should only be affected in its association with MMS2 but not with RAD5.
The results of the two-hybrid analysis are shown in Fig. 2B.
As expected, mutant UBC13(E55A) was found to associate with RAD5, but
interaction with MMS2 was abolished. In contrast, all other mutants
were capable of association with MMS2 but had lost their ability to
interact with RAD5. These results indicate that amino acids critical
for UBC13-RAD5 contacts reside within the N-terminal helix of UBC13 (Lys6 and Lys10) as well as the loops L1
(Met64) and L2 (Ser96) (Fig. 1B).
Thus, the UBC13 interface to the RAD5 RING finger closely resembles the
corresponding interfaces of other E2 enzymes with their respective
ubiquitin-protein ligases.
Consequences of Interface Mutations for in Vivo Function--
To
examine the effects of the different contact site mutations on the
ability of RAD5 and UBC13 to cooperate in DNA damage repair in
vivo, the mutant genes were integrated into the genome and
expressed under the control of their own promoters in the respective
deletion strains. Fig. 3 shows a
comparison of the UV sensitivities of representative mutants. Mutant
C914S, in which one of the Zn2+-coordinating cysteines is
replaced by serine, showed by far the largest effect on cell survival;
in fact, its UV sensitivity was more severe than that of the
ubc13 deletion, indicating that a structural perturbation of
the RING domain affects more aspects of RAD5 function than solely its
cooperation with UBC13 (Fig. 3A). Nevertheless, C914S was
epistatic to ubc13, because deletion of the E2 in this
mutant had no additional effect on UV sensitivity, suggesting that
UBC13 function in DNA repair entirely depends on the integrity of the
RAD5 RING domain. Yet, this mutant was less sensitive to UV irradiation
than a rad5 deletion; thus, RAD5 must have an additional
role in DNA repair that is independent of its RING finger. Mutant I916A
displayed a UV sensitivity very similar to that of the ubc13
deletion (Fig. 3A). The fact that the double mutant
ubc13 rad5(I916A) showed no further increase in UV
sensitivity indicates that mutation of this residue apparently eliminates all aspects of RAD5 functions that involve a cooperation with UBC13 but leave all of its UBC13-independent functions intact. Mutation D923A had no influence on the UV sensitivity of the respective strain, again confirming that this residue is not situated on a surface
critical for function in DNA repair (for the sake of clarity, this
curve is omitted from Fig. 3 and shown in supplementary Fig.
1A). Surprisingly, mutations E943A, Y944A, and N959A only marginally affected UV sensitivity, even though in both Y944A and N959A
residues critical for UBC13 interaction were involved (Fig.
3B and supplementary Fig. 1A). In each case,
however, ubc13 was epistatic to the rad5 point
mutation, because the respective double mutants had UV sensitivities
indistinguishable from the ubc13 single mutant.
Parallel to the RAD5 constructs, the UBC13 contact site mutants were
examined in UV sensitivity assays. Fig. 3C shows a situation very similar to that of the RAD5(Y944A) and (N959A) mutations: all
mutants affecting the RAD5 interface in the two-hybrid system (K6E,
K10E, M64A, and S96A) were only slightly more UV-sensitive than the
wt strain (for the sake of clarity, survival curves of K6E, K10E, and M64A, which overlap with that of S96A, have again been omitted and are shown in supplementary Fig. 1B).
Remarkably, even UBC13(E55A), which had previously been shown to be
defective in DNA repair when present on a centromeric vector (43),
displayed the same subtle effect as the other interface mutants when
integrated into the genome under the control of the native promoter
(Fig. 3C). In contrast, mutant UBC13(C87S), which lacks the
active site cysteine critical for catalytic activity, was as sensitive
as the ubc13 deletion.
DNA Repair Capacities Depend on the Expression Levels of
UBC13--
These results suggested that mutants in which
protein-protein contacts are compromised may display defects whose
magnitude depends on the genetic context, whereas those changes that
directly affect the catalytic center or the structural integrity of the proteins would lead to a permanent loss of function. To examine this
possibility, the UBC13 mutants were expressed from a centromeric vector
under the control of the regulatable MET3 promoter. On synthetic selective medium, which contains 0.67 mM
methionine, this promoter should be partially repressed (46). When UV
sensitivities were compared in this context, a striking difference
between wt UBC13 and the interface mutants became apparent;
in fact, here all contact site mutants, including E55A, were as
sensitive as the catalytic site mutant and the ubc13
deletion (Fig. 3D and supplementary Fig. 1C).
Fig. 3E shows that expression from the MET3
promoter resulted in significantly lower protein levels than expression
from the native UBC13 promoter even in the absence of damage-induced
up-regulation. These findings imply that the UBC13 interaction mutants
might only show significant defects in DNA repair when protein levels
are limiting, whereas under physiological conditions, when
transcription of the UBC13 promoter can be induced by DNA damage,
increased expression levels can overcome the weakened interaction with
RAD5 or MMS2.
To test this notion more rigorously, UV sensitivity of mutants
representative for the RAD5 contact site (S96A), the MMS2 interface (E55A) as well as the catalytic center (C87S) were compared in UV
gradient assays on plates of defined methionine concentration that
exploited the entire range of the MET3 promoter, reaching from full induction at 0 mM methionine to full repression
at 2 mM (Fig. 4A).
In parallel, protein expression under both extremes was examined (Fig.
4B). In the absence of methionine the MET3 promoter afforded an expression level of UBC13 higher than that of the
native wt strain; under these conditions, the unmutated UBC13 as well as the interaction mutants displayed wt UV
sensitivity, and only the catalytic site mutant C87S was deficient in
DNA repair. At 2 mM methionine, where no expression of
UBC13 from the MET3 promoter was detectable, mutants S96A
and E55A showed UV sensitivities equal to that of C87S, whereas for the
unmutated UBC13 under control of the MET3 promoter partial
sensitivity in between that of the true wt and the mutants
was observed. Intermediate phenotypes were observed at 0.33 and 0.67 mM methionine (data not shown). Thus, these results support
the notion that weakened protein-protein interactions lead to
phenotypically visible repair defects only under circumstances where
protein levels are limiting.
Determination of Dissociation Constants of UBC13-MMS2
Dimers--
The variable UV sensitivities of UBC13(E55A) and the
mutants unable to interact with RAD5 stand in contrast to the mutant MMS2(F8A). This mutation had previously been demonstrated to disrupt the interaction between MMS2 and UBC13 in a manner similar to UBC13(E55A), and a similar loss of in vivo function was
shown by means of UV sensitivity assays (43). Unlike the UBC13
interaction mutants, however, MMS2(F8A) proved to be a complete
loss-of-function mutant in this study even when integrated into the
genome under the control of its native promoter (data not shown).
Considering these differences, it was of interest to determine to what
degree the interactions involving UBC13 were weakened in the individual contact mutants. Thus, association between UBC13 and MMS2 was quantified for a number of combinations involving wt or
mutant proteins using surface plasmon resonance (Biacore) technology. Recombinant UBC13 was produced as a GST fusion protein for
immobilization on the sensor surface, whereas MMS2 was expressed in a
self-cleavable intein fusion system (see supplementary Fig.
2A). The dimer resulting from the wt constructs
was catalytically active (see below). Initial experiments showed that
binding and dissociation of UBC13 and MMS2 occurred with kinetics too
fast to be accurately resolved by the instrument. Therefore,
dissociation constants (KD) were determined from a
plot of the signal at equilibrium against the concentration of the
soluble binding partner (see supplementary Fig. 2, B-F).
The results for the different combinations of wt and mutant
proteins are shown in Table I. To confirm
that the value of KD = 3.1 × 10 Catalytic Activities Correlate with Affinities and in Vivo
Function--
If the repair defects observed in vivo are
really caused by the differential affinities of the proteins, then this
correlation should also be reflected by the catalytic activities of the
respective combinations of mutants. To test this model,
RAD5-independent multiubiquitin chain synthesis was assayed in
vitro using the same set of recombinant proteins as in the
interaction analysis. Ubiquitin polymerization was monitored over a
time course of 4 h at 30 °C, using two different E2
concentrations. Under these conditions the wt combination of
GST-UBC13 and MMS2 displayed detectable chain synthesis activity when
present at 0.5 µM, whereas at 5.0 µM E2
free ubiquitin was largely consumed at the end of the incubation period
(Fig. 5A). Similar activities
were obtained for pairs of proteins bearing other combinations of GST
and His6 tags or those produced in the native
form,3 confirming that
N-terminal fusions to either protein influence neither mutual
interaction nor catalytic activity. As expected, UBC13(S96A) showed
wt levels of ubiquitin chain synthesis in combination with
wt MMS2 (Fig. 5B), confirming that the repair
defect observed in vivo was not due to a reduction in
catalytic activity of the protein itself but rather to a weakened
interaction with RAD5. Mutants K6E, K10E, and M64A were also found
catalytically active as GST or His6 fusion
proteins.2 In confirmation of previous results (43),
MMS2(F8A) in combination with wt GST-UBC13 produced no
multiubiquitin chains except for ubiquitin dimers at high E2
concentration, (Fig. 5C). The high molecular weight species
reactive to the anti-ubiquitin antibody observed at 5.0 µM E2 result from auto-ubiquitylation of GST-UBC13 (47),
a side reaction that occurs even in the absence of MMS2.2
Interestingly, UBC13(E55A) in combination with wt MMS2
showed no detectable chain synthesis at low concentration but showed an
activity comparable with the wt combination at 5 µM E2 (Fig. 5D). These results indicate that
with respect to catalytic activity UBC13(E55A) is able to overcome the
reduced affinity for MMS2 at high protein concentration, a notion that
can explain the dependence of its in vivo repair phenotype
on the expression level.
The RAD5-UBC13 Interface Resembles Those of Typical E2-E3
Pairs--
This study sheds light on the nature of the interfaces
within the complex between the RING finger protein RAD5 and the dimeric ubiquitin-conjugating enzyme UBC13-MMS2. Site-directed mutagenesis has
demonstrated that the UBC13 subunit utilizes the same structural elements for contact with the RING finger as many monomeric E2s. In the
c-Cbl-UbcH7 structure, the critical residues are Phe63 in
the L1 loop as well as Pro97 and Ala98 in L2
(Ref. 15 and Fig. 1). The same positions were found to participate in
binding of UbcM4 to a series of RING finger proteins (48), and even the
interaction of UbcH7 with the structurally unrelated HECT-type E3,
E6-AP, involves identical contacts (44). Phe63 had earlier
been shown to be of particular importance for E2-E3 specificity (49).
In UBC13, the corresponding position is occupied by Met64,
and mutation to alanine similarly abolished interaction with RAD5.
Pro97 in L2 was not mutated in this study to avoid a
perturbation of the E2 backbone; however, the neighboring residue,
Ser96, was shown to have significant impact on RAD5
binding. Thus, it appears that the identities of the amino acids at the
tip of the L1 and L2 loops confer specificity to the E2-RING finger
interaction in the case of UBC13 as well. Based on the observation that
charged amino acids in the N-terminal
The surface of the RING domain involved in the contact to the E2 forms
a shallow groove in both c-Cbl and CNOT4 (15-17). Centrally positioned
on this surface are several hydrophobic residues, Ile383
and Trp408 in c-Cbl and Leu16 and
Ile45 in CNOT4, mutations of which were shown to abolish E2
binding (12, 16). Similarly, in the BRCA1 RING finger, which was
recently found to possess ubiquitin ligase activity, mutation of
Leu51 (corresponding to c-Cbl Trp408) to
alanine resulted in a loss of E2 binding and catalytic activity (18).
In RAD5, the corresponding residues, Ile914 and
Tyr944, were found to fulfill similar functions, although
mutations to alanine affected in vivo function of the
protein to different degrees, with Ile914 having by far
greater influence on the observed phenotype than Tyr944. In
contrast, the neighboring Glu943, whose analog in c-Cbl is
likewise involved in E2 binding, had no effect on association with
UBC13. These findings demonstrate that although the E2-RING finger
interface in the RAD5-UBC13 complex is structured similarly to those of
several well studied E2-E3 pairs, subtle differences in the
contributions of individual residues are likely to convey an exclusive
specificity to the interaction. The overall conservation of the contact
surfaces, however, strongly implies a productive E2-E3 relationship for
the RAD5-UBC13 interaction, thus supporting a model that attributes
ubiquitin ligase activity to the RAD5 protein.
Strong Interactions Are Dispensable for Cooperation between RAD5,
UBC13, and MMS2 in Vivo--
Perhaps the most intriguing result of
this study is the fact that some of the contact site mutations in RAD5
and UBC13 have surprisingly small effects on the UV sensitivities of
the resulting strains. If a high affinity association were necessary
for cooperation between the UBC13-MMS2 dimer and RAD5, loss of this
interaction by mutation of either RAD5 or UBC13 should result in the
same phenotype as that of a ubc13 deletion, a condition that
was observed only for the RAD5(I916A) mutant. The absence of a visible
phenotype would thus imply that the components could function in
isolation and would not have to interact in vivo. However,
this scenario seems unlikely, because all aspects of UBC13 and MMS2
function reported to date depend on the presence of the RAD5 protein
(36, 41, 53, 54), and a recruitment of the E2 to chromatin by RAD5 has
been demonstrated (36). Moreover, if RAD5 indeed functions as a
ubiquitin ligase, contact with its cognate E2 should be necessary for
ubiquitylation. Thus, it appears more likely that those mutations with
minor phenotypic consequences cause only a partial weakening of the
E2-RING finger interaction, which would lead to a loss of signal in the
(qualitative) two-hybrid system but may not completely abolish
association in vivo. In addition, the affinity of an E2 to
its cognate E3 as measured in isolation may not always correspond to
the situation during the ubiquitylation reaction, because ubiquitin, substrate, or product binding may have consequences for the E2-E3 interaction. Accordingly, a number of precedents for productive low
affinity interactions between E2s and RING finger E3s have been
described (13), and in the case of the E3 UBR1, which contacts its cognate E2 RAD6 by means of a domain distinct from the RING finger,
this interaction can be completely disrupted without significant loss
of ubiquitin ligase activity (12-14). In contrast, mutation of the
UBR1 RING finger abolishes E3 function, implying that an intact RING
domain is required for catalytic activity rather than merely for the
recruitment of the E2. The same appears to apply to the RAD5 RING
finger, where perturbation of the domain structure by the C914S mutant
had much more severe consequences than mutation of individual surface residues.
The concept of weak but productive interactions in the RAD5 and UBC13
contact mutants raises the question of how much the affinity in this
complex can be reduced without significant loss of cooperation. I have
started to approach this problem using the association within the
UBC13-MMS2 dimer as a model system, because this complex is readily
accessible for in vitro analysis. VanDemark et
al. (43) have demonstrated that complex formation is required for
ubiquitin polymerization by UBC13-MMS in vitro, because the
MMS2(F8A) mutation, which abolishes interaction with UBC13, was
severely compromised in a conjugation assay. Mutation of
Glu55 to alanine in UBC13 similarly reduced complex
formation, and both mutants were found inactive in vivo
(43). I have now presented evidence that in contrast to MMS2(F8A) the
defect of the UBC13(E55A) mutant can be overcome in vivo as
well as in vitro by means of mass action via an
increase in protein concentration. Whether the more severe phenotype of
UBC13(E55A) observed by VanDemark et al. (43) is
attributable to differences in strain background or details of promoter
size or vector backbone remains unresolved. Consistent with the
differing phenotypes observed in this study, however, the extent of
interaction defects differs dramatically between UBC13(E55A) and
MMS2(F8A); whereas the former causes an ~60-fold reduction in
affinity, the latter mutation was found to reduce binding by more than
3 orders of magnitude. Based on the fact that the UBC13(E55A) displayed
a phenotype identical to those mutants defective in their interaction
with RAD5, the values obtained for interaction with MMS2 can be used as
a benchmark in the analysis of the E2-RING finger contacts, which are
likely to be of a similar order of magnitude, given comparable
concentrations of the individual factors in the nucleus. This
assumption has yet to be confirmed but appears probable based on the
notion that overall RAD5 levels are significantly lower than those of
UBC13 or MMS2,2 but the bulk of UBC13 and MMS2 resides
outside the nucleus even under conditions of DNA damage (36).
Another notable property of the UBC13-MMS2 interaction is the extremely
fast kinetics of association and dissociation that suggests a highly
dynamic interaction rather than a permanent complex. NMR analysis of
complex formation between UbcH5 and the CNOT4 RING finger implies a
similar mode of interaction (16). Several interpretations can be
envisioned to account for this kinetic lability. On one hand, fast
dissociation rates may favor a rapid equilibrium between alternative
complexes in the postreplication repair system as predicted earlier
(36). On the other hand, the dynamic nature of the complex may be
required for catalytic activity itself, e.g. to allow
product dissociation or to facilitate activation by E1. In fact,
McKenna et al. (55) argue that human UBC13 becomes less
accessible to E1 when complexed to MMS2. Thus, dissociation of the
UBC13-MMS2 complex might actually occur following each round of catalysis.
Additional Functions of RAD5 in DNA Repair--
The fact that a
rad5 deletion causes a much more severe UV sensitivity than
a ubc13 or mms2 null mutant (36, 54) implies that
RAD5 must have an additional role in DNA damage repair independent of
its cooperation with UBC13 and MMS2. The RAD5 mutant analysis presented
here allows the differentiation between RING
finger-dependent and -independent aspects of RAD5 function.
Mutation I916A exhibits a phenotype identical to and epistatic with the
ubc13 deletion; this implies a complete loss of all activity
related to cooperation with UBC13-MMS2, i.e.
ubiquitin-protein ligase function. Yet, the C914S mutation confers a
stronger phenotype than I916A, suggesting that the RING finger itself
may contribute to UBC13-independent aspects of RAD5 function.
Alternatively, disruption of the RING finger by removing one of the
Zn2+ coordination sites may perturb the structure of the
helicase-like domain into which the RING finger is embedded, which
might result in a stronger phenotype than the exchange of a surface
residue within the RING. Another explanation would be a decreased
protein concentration of C914S; this seems unlikely, however, because preliminary experiments indicate that upon overexpression the mutant
exhibits a stability similar to that of the wt
protein.2
Even the C914S mutant is not fully devoid of activity, because its UV
sensitivity is still less severe than that of the rad5 deletion. In light of these findings it is noteworthy that Martini et al. (56) recently identified a histone H2B mutant,
htb1-3, that displayed a UV sensitivity hypostatic to RAD5
but additive with respect to a ubc13 mutation. Considering
the DNA-dependent ATPase activity of RAD5 (32) and its
homology to members of the SNF/SWI family, which includes helicases and
chromatin remodeling factors (57, 58), it is attractive to speculate
that beyond its ubiquitin ligase function RAD5 could be involved in the
removal or repositioning of nucleosomes during the recession of the
replication fork upon the encounter of a lesion in the template strand,
thereby facilitating the strand switching process proposed to initiate postreplication repair (21, 29-31). Biochemical analyses will be
necessary for a conclusive demonstration of RAD5 ubiquitin-protein ligase activity and for the investigation of additional activities independent of ubiquitylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B signaling
(26). In yeast and mammals, the only enzymatic activity demonstrated so far to catalyze the assembly of Lys63-linked ubiquitin
chains resides in the heterodimeric complex of UBC13 and MMS2, a
genuine UBC and a structurally related ubiquitin-conjugating enzyme
variant (27). The function of Lys63-linked ubiquitin chains
in DNA damage repair has been attributed to a participation of UBC13
and MMS2 in the postreplication repair pathway (27, 28), which confers
damage tolerance and ensures the completion of genome duplication in
situations where lesions in the template strand cause a stalling of the
replication machinery (21, 29-31). The principal mediator of
postreplication repair is RAD6 (32), which encodes another UBC (33). It
is believed to be targeted to sites of damage by the DNA-binding RING
finger protein RAD18 (34). An error-free subpathway within the RAD6 system is mediated by another chromatin-associated RING finger protein,
the SWI/SNF homolog RAD5 (35). I have previously shown that the
function of UBC13 and MMS2 in the RAD6 pathway in yeast is mediated by
RAD5, which contacts UBC13 through its RING domain and recruits the E2
heterodimer to the chromatin in response to DNA damage (36). Through
its association with the RAD6-RAD18 dimer, RAD5 thus coordinates the
assembly of two different E2s on damaged chromatin, suggesting a
cooperation of RAD6 and the UBC13-MMS2 complex with the RING finger
proteins RAD18 and RAD5 in ubiquitin conjugation (36). In confirmation
of this model it was recently shown that proliferating cell nuclear
antigen, a processivity factor for a number of DNA polymerases
dedicated to replication as well as repair, is modified by
Lys63-linked ubiquitin chains in response to DNA damage
(37). This modification depends on the presence of RAD5, UBC13 and
MMS2, whereas RAD6 and RAD18 in the absence of the former afford only mono-ubiquitylation (37). Thus, it appears that the UBC13-MMS2-RAD5 assembly indeed functions as a genuine E2-E3 complex for the assembly of Lys63-linked multiubiquitin chains. Support for this
notion is presented here by a genetic and biochemical analysis of the
protein-protein interactions within this E2-RING finger complex and
their consequences for cooperation between RAD5 and the UBC13-MMS2
dimer in DNA damage repair.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 s
1. Selective medium was used
before and after irradiation for strains bearing centromeric vectors.
-mercaptoethanol treatment, and the total
protein was precipitated with trichloroacetic acid as described (42).
Aliquots were analyzed on 15% SDS-polyacrylamide gels, followed by
Western blots using an affinity-purified anti-UBC13 antibody (see below).
-D-thiogalactopyranoside for 6 h at
28 °C. Purifications were achieved in a single step by affinity
chromatography on the appropriate columns (glutathione-Sepharose
(Amersham Biosciences), nickel-nitrilotriacetic acid resin (Novagen or
Qiagen), or chitin beads (New England Biolabs) for GST,
His6, or chitin-binding domain-intein fusions,
respectively) according to the manufacturers' recommendations. Purified proteins were dialyzed against 25 mM Tris-HCl, pH
7.5, 50 mM NaCl, 0.5 mM EDTA and stored frozen
at
80 °C in the presence of 10% (v/v) glycerol. The protein
concentrations were determined by absorbance at 280 nm based on
calculated extinction coefficients. Polyclonal antibodies against the
His6-UBC13 protein were raised in rabbit and purified by
affinity chromatography on CH Sepharose 4B (Amersham Biosciences)
covalently derivatized with GST-UBC13.
2
values were below 10 and the residuals did not exceed a few RU.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (43K):
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Fig. 1.
Design of RAD5 and UBC13 interface
mutants. The filled circles above the c-Cbl, CNOT4, and
UbcH7 sequences highlight residues that were shown to be located at the
RING-E2 interface based on the x-ray structure (c-Cbl and UbcH7) or
chemical shift changes (CNOT4). The filled circles above the
RAD5 and UBC13 sequences indicate the positions for site-directed
mutagenesis. The open circles represent the locations of
control mutations. A, sequence alignment of the RAD5 RING
finger with the RING domains of c-Cbl and CNOT4. Cysteine and histidine
residues involved in zinc coordination are shown in bold
type. B, sequence alignment of UBC13 with the
respective binding partners of c-Cbl and CNOT4, UbcH7, and UbcH5. The
catalytic cysteine residue is shown in bold type. Secondary
structure elements are indicated above the alignment: H,
-helix; S,
-sheet; L, loops according to
Ref. 44.
-helix
(H1) as well as residues in the loops between
-sheets S3 and S4 as
well as
-helices H2 and H3 (Ref. 15 and Fig. 1B). The
same structural elements are used in the binding of UbcH7 to a HECT
type E3 (44). The crystal structures of the yeast and the human
UBC13-MMS2 dimers revealed that the corresponding residues in UBC13 are
accessible, and the presence of MMS2 would permit simultaneous contacts
to the RAD5 RING finger (43, 45). Therefore, mutations in UBC13 were
designed at the following positions: K6E and K10E in helix H1 and M64A
and S96A in the loops. Mutation of Pro97, although more
closely representative of the respective contact site in UbcH7 than
Ser96, was avoided to exclude a perturbation of the overall
protein structure. Mutation E55A was introduced as a control, because this residue had previously been shown to reside in the interface to
MMS2 (43, 45) and should therefore have no effect on interaction with
the RAD5 RING finger.
View larger version (47K):
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Fig. 2.
Effect of point mutations on interaction
between RAD5 and UBC13. Fusions to the GAL4 activation
(AD) or DNA-binding domain (BD) were examined in
the two-hybrid system for their ability to activate the HIS3
reporter gene of the tester strain PJ69-4A, as indicated by growth on
selective ( His) medium. A, interactions of RAD5 RING
finger mutants with wt RAD5 and UBC13. B,
interactions of UBC13 mutants with MMS2 and RAD5.
View larger version (29K):
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Fig. 3.
Effect of representative mutations in RAD5
and UBC13 and expression level on UV sensitivity. A-D,
survival after irradiation (254 nm) is plotted against the applied UV
dosage. Expression vectors used are indicated on each panel. Those
mutants mentioned in the text but not shown in this figure can be found
in supplementary Fig. 1. A and B represent an
analysis of RAD5 constructs in the rad5 (filled
symbols) and the rad5 ubc13 background (open
symbols). A, squares, RAD5;
circles, C914S; triangles, I916A;
diamonds, empty vector. B, squares,
RAD5; circles, E943A; triangles, Y944A;
diamonds, empty vector. C and D show
the survival curves of UBC13 mutants in two different vectors in a
ubc13 strain. Filled squares, UBC13; open
squares, empty vector; filled circles, E55A;
filled triangles, S96A; open triangles, C87A.
E, expression levels of UBC13 and its mutants in the two
different vector backgrounds. UBC13 was detected by Western blot in
whole cell lysates prepared from cultures of equal densities. Shown for
comparison is the endogenous UBC13 of an unmodified wt
strain. Note that the bottom panel results from more
concentrated extracts. The aberrant migration of UBC13(E55A) has been
observed before (43).
View larger version (71K):
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Fig. 4.
Correlation of UBC13 expression level with UV
sensitivity. A, gradient plate assays. wt
cells and the indicated UBC13 mutants in YCp111-PMET3 grown
in the presence of different methionine concentrations were streaked
out on plates and subjected to a gradient of UV irradiation (0-3 min).
B, UBC13 protein levels resulting from the
YCp111-PMET3 constructs were examined after growth at the
indicated methionine concentrations and compared with those of the
wt strain. Lane 1, native wt;
lane 2, UBC13; lane 3, E55A; lane 4,
S96A; lane 5, C87S.
7
M for the wt-wt combination reflected
the physiological situation as closely as possible and to exclude an
effect of the GST moiety fused to UBC13, interaction was also measured
for a combination of proteins where MMS2 was fused to GST and UBC13 was
produced in the native form without any tag. The KD
value obtained for this pair of proteins was similar to that of the
reverse combination (Table I). As expected, mutation UBC13(S96A), which
should only affect binding to the RING finger of RAD5, had no effect on
the affinity for MMS2. In contrast, mutation UBC13(E55A) reduced the affinity for MMS2 by ~60-fold. Association of MMS2(F8A) with UBC13 was detectable only at high MMS2 concentrations, with a
KD of 1.5 × 10
3 M.
Because of the weak signals involved, this value should be viewed as an
estimation. These values imply that a moderate reduction in affinity as
observed in the UBC13(E55A) mutant can be partially overcome in
vivo, whereas a more severe reduction, as exhibited by the
MMS2(F8A) mutant, leads to a complete loss of function in DNA
repair.
Effects of point mutations on the affinities between UBC13 and MMS2
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Fig. 5.
Catalytic activities of UBC13 and MMS2
mutants. Formation of multiubiquitin chains by in vitro
polymerization reactions was analyzed by gel electrophoresis and
anti-ubiquitin blots of samples taken at the indicated time points.
A, GST-UBC13 and MMS2; B, GST-UBC13(S96A) and
MMS2; C, GST-UBC13 and MMS2(F8A); D,
GST-UBC13(E55A) and MMS2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix contribute to c-Cbl
binding in UbcH7, two lysine residues, Lys6 and
Lys10, were also examined in UBC13, and both were found to
be important for RAD5 binding. The N-terminal helix contributes to E3
binding to different extents in a number of other UBCs, such as RAD6 in complex with both of its known interaction partners, UBR1 (50, 51) and
RAD18 (52), as well as UbcM4 (48). These interactions, however, often
involve regions of the E3 outside the RING domain (14, 15, 52).
Similarly, I have previously shown that the RAD5 RING finger alone
is necessary but not sufficient for interaction with UBC13, suggesting
that the UBC13 H1 helix may contact regions of RAD5 N-terminal of the
RING domain, which might contribute to specificity.
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ACKNOWLEDGEMENTS |
---|
I thank Margret Ludwig for excellent technical assistance, Thomas Albert for the gift of untagged UBC13, Kristine Schmidt and Thomas Albert for help with construction of UBC13 mutants, and all of the laboratory members as well as Charles Cho for helpful discussions and valuable comments on the manuscript. Regine Kahmann is acknowledged for generous support.
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FOOTNOTES |
---|
* This work was supported by Grant UL188/1-2 from the Deutsche Forschungsgemeinschaft, the BioFuture program of the German Ministry for Education and Research, and a Young Investigator Grant from the German Israeli Foundation for Scientific Research and Development.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains supplemental figures.
To whom correspondence should be addressed. Tel.:
49-6421-178601; Fax: 49-6421-178609; E-mail:
hulrich@staff.uni-marburg.de.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M212195200
2 H. D. Ulrich, unpublished observation.
3 T. K. Albert and H. D. Ulrich, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: E1, ubiquitin-activating enzyme; E2 or UBC, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; wt, wild type; ORF, open reading frame; GST, glutathione S-transferase; RU, resonance units.
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