Mre11 Dimers Coordinate DNA End
Bridging and Nuclease Processing
in Double-Strand-Break Repair
R. Scott Williams,1,3,6Gabriel Moncalian,1,3,6,8Jessica S. Williams,1Yoshiki Yamada,1Oliver Limbo,1David S. Shin,1,3
Lynda M. Groocock,1Dana Cahill,4Chiharu Hitomi,1,3Grant Guenther,1Davide Moiani,1,3James P. Carney,4,7
Paul Russell,1,2,* and John A. Tainer1,3,5,*
1Department of Molecular Biology
2Department of Cell Biology
3Skaggs Institute for Chemical Biology
The Scripps Research Institute, 10550 North Torrey Pines Road, MB4, La Jolla, CA 92037, USA
4The Radiation Oncology Research Laboratory, Department of Radiation Oncology, University of Maryland School of Medicine,
Baltimore, MD 21201, USA
5Life Sciences Division, Department of Molecular Biology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
6These authors contributed equally to this work
7Present address: Battelle, Edgewood Chemical Biological Center, AMSRD-ECB-RT-BM, Bldg. E3831, 5183 Blackhawk Road,
Aberdeen Proving Ground, MD 21010, USA
8Present address: Instituto de Biomedicina y Biotecnologı ´a de Cantabria, 39011 Santander, Spain
*Correspondence: email@example.com (P.R.), firstname.lastname@example.org (J.A.T.)
Mre11 forms the core of the multifunctional Mre11-
Rad50-Nbs1 (MRN) complex that detects DNA
double-strand breaks (DSBs), activates the ATM
checkpoint kinase, and initiates homologous recom-
bination (HR) repair of DSBs. To define the roles of
Mre11 in both DNA bridging and nucleolytic process-
ing during initiation of DSB repair, we combined
tures of Pyrococcus furiosus Mre11 dimers bound to
DNA with mutational analyses of fission yeast Mre11.
The Mre11 dimer adopts a four-lobed U-shaped
structure that is critical for proper MRN complex
ther, mutations blocking Mre11 endonuclease activ-
ity impair cell survival after DSB induction without
pendant recruitment of Ctp1, an HR factor, to DSBs.
These results show how Mre11 dimerization and nu-
clease activities initiate repair of DSBs and collapsed
replicationforks, aswellas provide amolecular foun-
dation for understanding cancer-causing Mre11 mu-
tations in ataxia telangiectasia-like disorder (ATLD).
DNA double-strand breaks (DSBs) are highly cytotoxic and
genome-destabilizing DNA lesions. Mre11, the core subunit of
the Mre11-Rad50-Nbs1 complex (Mre11 complex or MRN),
plays central roles in DSB detection and repair. The importance
of the Mre11 complex has been established in Saccharomyces
cerevisiae and Schizosaccharomyces pombe, where it is critical
for survival of DSBs induced by ionizing radiation (IR) and other
genotoxins, aswellasin humans,wherehypomorphic mutations
of MRE11 and NBS1 cause cancer-prone syndromes of ataxia
telangiectasia-like disorder (ATLD) and Nijmegen breakage syn-
drome (NBS), respectively (Chahwan et al., 2003; Williams et al.,
2007). ATLD and NBS cells exhibit cell cycle checkpoint defects,
genome instability, and IR hypersensitivity (Carney et al., 1998;
Stewart et al., 1999).
As a DSB ‘‘first responder,’’ MRN recruits the ATM checkpoint
kinase (yeast Tel1) (Lee and Paull, 2005) through binding to Nbs1
cade leading to cell cycle arrest and checkpoint responses key
to genome integrity in humans, as evident from severe cancer
predisposition and IR-sensitive phenotypes of ATM null patients
(Shiloh, 2003). However, Mre11 complex subunits are essential
for organism and cell viability in mammals, whereas ATM is
not, so ATM recruitment and activation is only one of the impor-
tant Mre11 complex functions.
One of the most interesting but least understood Mre11 com-
Atomic force (AFM) (Chen et al., 2001; de Jager et al., 2001;
ing (Hopfner et al., 2001; Hopfner et al., 2002) show that MRN
structurally segments into a globular DNA-binding head,
elongated mobile Rad50 coiled coils, and distal Rad50 hook
domain. EM and AFM indicate DNA-bound heterotetrameric
Simultaneous DNA binding within one M2R2DNA-binding head
drives short-range bridging of DNA ends to within 100 A˚of one
another (Chen et al., 2001; Hopfner et al., 2002). Alternatively, re-
versible Zn2+-dependant Rad50 hook domain assembly erects
Cell 135, 97–109, October 3, 2008 ª2008 Elsevier Inc. 97
Figure 1. Mre11 X-Ray Structures for Synaptic and Branched DNA Complexes
(A and B) Mre11 complex association with synaptic (A) and branched (B) DNA ends shown schematically.
(C) Synaptic DNA end complex experimental electron density. The 2.7 A˚resolution composite simulated annealing 2Fo-Fc electron density map contoured at
0.8s shows the bound DNA (yellow) overlaid upon Mre11 dimer density (gray).
98 Cell 135, 97–109, October 3, 2008 ª2008 Elsevier Inc.
two-headed octameric (M2R2)2scaffolds to tether DNA chains up
suggest that the DNA-binding head bridges variable ends via the
Mre11 protein, which interacts with ssDNA, dsDNA, and forked
DNA structures (Trenz et al., 2006; Wen et al., 2008). However,
the basis for Mre11 complex DNA interactions is unknown
because of an absence of high-resolution structures for any of
the Mre11 complex components bound to DNA.
Anotherkey andpoorlyunderstood Mre11complexfunctionis
its role in nucleolytic processing of DNA ends. Mre11 has both
ssDNA endonuclease and 30-50exonuclease activities when as-
of DSBs in vivo (Williams et al., 2007). This resection generates
a 30ssDNA tail, which is critical for DSB repair by homologous
recombination (HR). Curiously, budding-yeast studies show
that mutations ablating Mre11 nuclease activities without desta-
bilizing the complex cause mild IR sensitivity yet have strong ef-
fects in meiosis and cleavage of hairpin structures. These find-
ings led to a prevailing view that Mre11 nuclease activity is not
needed for DSB repair except in situations involving blocked
DNA ends or unusual DNA structures (Bressan et al., 1998;
Krogh et al., 2005; Lewis et al., 2004). Yet, structural and bio-
chemical studies indicate that Mre11 is the core of the MRN
complex DNA-binding and -processing head, participating in
intermolecular interactions with itself, Rad50, Nbs1, and DNA
substrates (Williams et al., 2007).
Here, we integrate crystallographic, small-angle X-ray scatter-
ing (SAXS), and biochemical analyses of archaeal Mre11-DNA
complexes with genetics in S. pombe to dissect the basis
for Mre11 homodimerization, Mre11-DNA interactions, short-
range Mre11-complex DNA synapsis, and Mre11 nucleolytic
mechanisms. Mre11-DNA complex structures allow the identifi-
cation of single-site mutants that retain Mre11 fold and interac-
tions but ablate either exonuclease activity or both endonucle-
ase and exonuclease activities. Furthermore, phenotypic
analyses of single-site mutants in fission yeast establish that
Mre11 dimerization and endonuclease activities are required
for survival of DSBs. In agreement with characterizations of an
Mre11 nuclease-dead mouse cell line in an accompanying paper
(Buiset al., 2008), these findings show Mre11 nuclease activity is
critical for DSB repair in both fission yeast and mammals.
Mre11-DNA Complex Structure Determination
Because DSBs are induced by ionizing radiation and form when
DNA replication forks encounter lesions (Wyman and Kanaar,
2006), they contain scission products with varied 50or 30over-
hanging ends or ssDNA-dsDNA junctions generated by replica-
tion fork collapse. We therefore crystallized and determined
X-ray structures of the catalytically active phosphoesterase
domains (comprising the nuclease and nuclease-capping do-
mains, residues 1–342) of Pyrococcus furiosus Mre11 bound to
two distinct DNA oligonucleotide targets (see the Experimental
Procedures, Figure 1, and Figure S1 available online). The struc-
tures define two types of Mre11-DNA interactions: (1) a synaptic
complex mimicking DNA end joining of products from a dsDNA
scission event bearing a short 2 bp 30overhang (Figures 1A,
1C, and 1E) and (2) a branched DNA structure that might be
encountered by Mre11 at a collapsed fork (Figures 1B, 1D, and
1F). Two approaches promoted stabilization of nuclease-DNA
complexes. For the synaptic end complex, we used EDTA
to chelate divalent cations from the Mre11 active site. For
the branched DNA complex, we used an H85S-inactivating
active-site mutation (characterized herein) that does not alter
active-site Mn2+binding, Mre11 stability, or macromolecular
interactions. The synaptic end complex at 2.7 A˚and branched
complex at 2.2 A˚resolution were phased by molecular replace-
ment, fit to unbiased omit maps, and refined (Table S1).
Architecture of Mre11-DNA Complexes
To characterize Mre11-DNA assembly interfaces, we examined
crystal packing and noncrystallographic symmetry interactions
in the DNA-bound crystal forms. Both structures reveal a con-
served four-helix bundle Mre11-Mre11 homodimeric interface.
This interface at a two-fold axis in the C-centered orthorhombic
unit cell in the synaptic complex (Figure 1C) matched that at
the interface between noncrystallographic symmetry (NCS)-
related protomers in the branched DNA complex (Figure 1D).
A related interface in the DNA-free Mre11 structure was as-
sumed to reflect crystal packing (Hopfner et al., 2001). These
two DNA-bound structures imply that this is the biologically
relevant Mre11 dimer that interacts with diverse DNA substrates
The dimer interface between helicies aB and aC of the Mre11
nuclease domain buries ?1400 A˚2of solvent-accessible surface
area with van der Waals packing between hydrophobic residues
lix-bundle periphery is reinforced by a Lys62 to Asp100 intermo-
lecular salt bridge and capped near the basic DNA-binding cleft
by Arg55 side-chain hydrogen bonding to an opposing Arg55
or pseudo-two-fold (branched complex) axis. Structure-based
sequence alignments for human, yeast, and archaeal Mre11 ho-
mologs reveal that the high sequence identity among residues in
this dimer interface is paralleled only by conservation within the
five active-site nuclease phosphoesterase motifs (nuclease
motifs I–V) and DNA-recognition loops (RL1–RL6) (Figure S1).
(D) Branched DNA complex experimental electron density. The sigma-A-weighted 2.2 A˚2Fo-Fc model phased map (calculated prior to building the DNA chain) is
contoured at 1.0s.
(E) Dimeric Mre11 can align and tether two DNA ends. Upper and lower orthogonal views show the Mre11-DNA complex for synaptic DNA ends: Mre11 fold as
ribbons for helix and beta strand and asyellow backbone tubes withplanar bases for DNA.Two Mre11 subunits, whichare oriented about acrystallographictwo-
fold axis (dotted line), bind the terminal ends of the DNA substrates (yellow).
DNA substrate asymmetrically in one half of the Mre11 dimeric cleft, with both Mre11 subunits contributing to binding one dsDNA. The Mre11 dimer orients
around NCS related protomer contacts.
Cell 135, 97–109, October 3, 2008 ª2008 Elsevier Inc. 99
The U-shaped Mre11 dimer enables two subunits to simulta-
neously contact the dsDNA portions of the DNA substrates (Fig-
ures 1 and 2). DNA makes extensive contacts to both N-terminal
calcineurin-like nuclease domains and to a single C-terminal
mixed a-b fold nuclease-capping domain (Figures 1A, 1B, and
but not equivalent (Figures 2B and 2C). Six DNA-binding regions
provide 17 residues for extensive DNA contacts in cis and in
trans across the dimer. These six DNA-recognition loops (RL1–
RL6) converge into a contiguous DNA interaction surface (Fig-
ures 2B–2D). DNA binding is mediated entirely by minor groove
contacts to the sugar-phosphate backbone, explaining how
Mre11 acts as a universal DNA-processing enzyme (Figure 2E).
For the large protein surface area (1525 A˚2) buried in the DNA in-
terface, there is ?2:1 (500:275 A˚2buried protein surface) distri-
bution of DNA-binding contacts for each subunit (Figure 2E).
Mre11 dimerization is therefore key to efficient DNA binding.
Bound dsDNA is B formwith key deviations in overall and local
helical parameters. Structural overlays of the synaptic complex
DNA with a B-DNA template and analyses of helical parameters
show that phosphate backbone and nucleotide conformation
for base pair positions distal from the nuclease motifs (A4:T15,
A5:T14, G6:C13, and C7:G12 base pairs; Figure 2D) have ideal-
to the active site (base pairs C3:G16, A2:T17, and C1:G18;
Figure 2D) show a protein-induced ?2 A˚minor groove widening
and helical axis distortion, coupled with a 10?bend toward the
major groove. Salt bridging and van der Waals contacts from
Lys327) engage the 50strand. The 30end direction is steered by
contacts from loops RL2 (His52, Arg55, and Ser57), RL1 (Tyr13
and Glu14) and RL3 (Asn89). In concert, RL3 Arg90 and three
groove, forming a wedge that redirects and distorts the duplex
backbone in the synaptic end and branching DNA complexes
(Figure 2D). Recognition and sculpting of the structurally mallea-
ble fray of the duplex end provides a probable determinant of
Mre11 DNA end recognition, DNA unwinding, 30-50nuclease,
All core DNA-binding elements are conserved in human,Xeno-
pus, S. cerevisiae, and S. pombe Mre11 homologs except RL3
end,whichliesadjacenttoRL3,may substituteforthese contacts
ential functional requirements of eukaryotic enzymes, whose
catalytic activities are modulated by Nbs1/Xrs2 (Lee et al., 2003;
Trujillo et al., 1998) and Ctp1/CtIP/Sae2 (Lengsfeld et al., 2007;
Limbo et al., 2007; Sartori et al., 2007).
DNA Synapsis and Branched DNA Binding
The Mre11-DNA structures show how Mre11 dictates multiple
DNA scaffolding functions. In the synaptic end complex, the
50 3 60 3 70 A˚binding cleft houses two opposed DNA ends
(Figures 1C and 1E). The DNA ends are tethered on a near paral-
lel trajectory, with an offset of about one duplex width when
viewed down the DNA helical axes. This linear bridging provides
a molecular mechanism with which DNA ends can be tethered
Figure 2. Mre11 Dimer-Mediated DNA-Binding Interactions
(A) Mre11 homodimeric interface. Stereo view shows the Mre11-Mre11 4-helix
bundle interactions at the DNA synaptic end complex crystallographic
(B) Dimeric Mre11 molecular binding surface (1.4 A˚probe) with dsDNA (yellow
tubes, bases, and surface) for the synaptic end DNA complex. Mre11:DNA
structures identify six Mre11 DNA-binding motifs: recognition loops RL1
(green),RL2 (purple), RL3 (turquoise), RL4 (pink), RL5 (dark blue),and RL6 (yel-
low). The minor groove wedge (green) is formed from RL1 residues E14, P10,
and H17 (colored molecular surfaces).
(C) Branched DNA complex with colored molecular surface for the RL1-RL6
DNA-binding motif interactions.
shows that all six Mre11 DNA-recognition loops engage the DNA minor groove
and phosphate backbone (yellow).
(E) Dimeric Mre11 molecular binding surface (1.4 A˚probe) for dsDNA (yellow
tubes, bases, and surface) including the roles of the two nuclease subunits
(light and dark blue) and the capping domain (gray) acting in binding the
DNA minor groove and backbone.
(F) Nuclease domain DNA-binding cleft (molecular surface colored by electro-
static potential from blue positive to red negative) showing that the branched
DNA 30tail is molded into a B-form conformation.
100 Cell 135, 97–109, October 3, 2008 ª2008 Elsevier Inc.
short distances (Chen et al., 2001; Hopfner et al., 2002) and ex-
plains Mre11 complex roles in DSB recognition and signaling
events (Limbo et al., 2007; Lisby et al., 2004).
In the branched complex, the substrate ssDNA-dsDNA junc-
tion binds at the nuclease-capping domain interface, implicating
the capping domain in ssDNA binding of branched DNA
substrates (Figures 2C and 2F). Unlike the synaptic complex,
the branched DNA binds asymmetrically across the dimer (Fig-
ure 1D). Capping domain movement occludes the second
DNA-binding site in the crystal. The four-nucleotide 30ssDNA
tail is diverted tangentially away from the dsDNA helical axis
and makes nonspecific contacts to a positively charged surface
Three conserved basic residues (Lys318, Arg332, and Lys327)
bind the phosphate backbone of the 30end and, intriguingly,
sculpt the last three 30nucleotides to adopt B form conformation
(Figure 2F). We therefore propose that capping domain-DNA
interactions can also participate in end-on dsDNA duplex
binding such that the two interaction surfaces would simulta-
neously accommodate branched architectures of partially repli-
cated collapsed replication forks. In combination, Mre11 dimer
and capping domain-DNA interactions shown in the crystal
structures appear suitable to dictate multiple DNA-tethering
Mre11 Dimerization Facilitates DNA Binding
To examine roles of Mre11 dimerization in DNA binding and
catalysis by the Mre11 complex in solution, we assessed
Mre11 shape and oligomeric state using analytical gel filtration
and SAXS (Putnam et al., 2007) (Figure 3 and Figure S2). Exper-
imental scattering curves for WT-Mre11 compared to calculated
scattering from monomeric or dimeric Mre11 model structures
reveals a close correspondence to the dimeric model and
a poor fit to the monomer profile (Figure 3E; Table S3). The cal-
culated radius of gyration (Rg) derived from SAXS Guinier analy-
sis (34.6 ± 0.02 A˚), the apparent molecular weight from analytical
gel filtration (?74 kDa), and bimodal shape and maximum parti-
cle dimension (Dmax= 120 A˚) found in the SAXS electron pair dis-
tribution function [P(r)] all agree with the bilobed Mre11 crystal
structures (Figures 3A and 3C, Figure S2, and Table S2). Align-
ment of the dimeric crystal structure with averaged molecular
envelopes from ab initio SAXS shape reconstruction further
Figure 3. Mre11 Dimerization Is Key to
Stable DNA Binding but Not Endonuclease
(A) Gel-filtration analysis of Mre11 dimerization.
Equivalent amounts (1.0 mg) of protein were
loaded onto a Tricorn Superdex200GL (GE Amer-
sham) column. Right: Coomassie-stained SDS-
PAGE of eluted fractions from the peaks between
13 mL and 18 mL.
(B) Mre11 dimer interface viewed in cross-section.
Leu61 and Leu97 side-chain pairs (gray and blue
subunits) pack together to form the hydrophobic
core of the four-helix bundle dimer interface.
(C) Wild-type Mre11 dimer and designed mutant
monomers characterized by small-angle X-ray
solution scattering (SAXS).SAXS electron pair dis-
tance distribution [P(r)] functions for WT Mre11
(blue) show that mutations in the Mre11 dimer
interface convert Mre11 to a smaller form.
(D) SAXS solution structure (transparent gray
envelope) for Mre11 overlaid with crystallographic
dimer structure (blue ribbons). Orthogonal views
of an averaged GASBOR SAXS ab initio solution
reconstruction envelope for WT Mre11 are aligned
with the Mre11 dimer structure, revealing that the
compact dimer interface is maintained in solution.
(E) Comparison of calculated SAXS curves for
Mre11 monomer (blue) and dimer (red) models
with experimental scattering for PfMre11 (black)
provides independent evidence that the wild-
type Mre11 is dimeric.
(F) EMSA DNA-binding analysis of pfMre11 dimer-
trations added are shown above wells. Left: EMSA
of Mre11 binding to 40-mer ssDNA. Right: EMSA
of Mre11 binding to 40 bp duplex DNA.
(G) Mre11 ssDNA endonuclease activity for dimer-
ization variants. Control shown is variant L61K
assayed at 37?C, where archaeal Mre11 nuclease
activity is negligible and contaminating nucleases
from E. coli would be active.
Cell 135, 97–109, October 3, 2008 ª2008 Elsevier Inc. 101
Figure 4. Differential Catalytic Requirements of Mre11 30-50Exonuclease and ssDNA Endonuclease Activities
(A) Proposed Mre11 30-50exonuclease catalytic cycle (see main text for details).
(B) The 30-50exonuclease activity for wild-type, H17A, H52S, and H85S Mre11 proteins assayed by monitoring of 2-aminopurine base release from a 2-AP-
containing dsDNA duplex. Error bars show ± 1 standard deviation.
(C) Mre11 DNA complexes superimposed by alignment of the nuclease domains. Core dsDNA-interacting regions for the synaptic complex (blue with green RL1
wedge residues) and branched end-binding complex (gray) are superimposed. A helical translocation of the bound DNA is coupled to capping domain rotation.
RL1 wedge His17 (green) advances 1.8 A˚into the minor groove.
(D) Structural basis for Mre11 30-50exonuclease reactions. The 50-dAMP (pink) position in the exonuclease product complex (RCSB ID:1II7) and observed 30
terminus in the synapticcomplex (gray) are shown relative to the proposed approach of the 30end (yellow). 30-50ssDNA exonucleolytic activity requires alignment
of the 30end by His52 and His85 (orange carbon tubes with blue nitrogen atoms).
102 Cell 135, 97–109, October 3, 2008 ª2008 Elsevier Inc.
reveals the close agreement of the solution structures with the
Mre11 crystallographic dimer assembly (Figure 3D).
To test whether the four-helix bundle assembly mediates self-
association, we introduced charged residue substitutions into
the Mre11 hydrophobic dimer interface. Four closely associated
conserved leucines are centered at the dimer core (Figure 3B).
Mre11 harboring mutations of these leucines to charged resi-
dues (Mre11-L61K or Mre11-L97D) retain normal thermostability
at 65?C but migrate as significantly smaller (?36 kDa apparent
molecular weight [MW]) protein species in gel filtration
(Figure 3A; Table S2). SAXS analysis shows that Mre11-L61K
and Mre11-L97D are converted to smaller forms with distinct
skewed monomodal P(r) functions and significantly smaller
Dmaxvalues (?85–90 A˚) than wild-type Mre11 (Figures 3C). Al-
though the gel-filtration analysis with proteins at ?12.5 mM
concentration shows effective disruption of stable Mre11 dimer-
ization by mutation of the hydrophobic interface, SAXS solution
scattering data for dimer mutants at high concentration (250 mM)
is best fit with a mixed monomer/dimer equilibrium model (Fig-
ure S2 and Table S3). Thus, dimerization is mediated via a con-
served hydrophobic four-helix bundle, and introduction of posi-
tive and negative charge into this interface disrupts the dimer.
To probe the functional importance of dimerization, we used
electrophoretic mobility shift assay (EMSA) analysis of DNA
binding and measured nucleolytic activities of dimer variants.
Wild-type Mre11 binds 40-mer ssDNA with high affinity (Kd=
170 nM) and has an ?6.5-fold lower affinity for 40 bp dsDNA
(Kd= 1.1 mM) (Figure 3F and Figure S3). In contrast, the mono-
meric Mre11-L61K and Mre11-L97D dimer mutants that lose
approximately one-third of their total DNA contact surface
show 30- to >50-fold decreased affinity for ssDNA, and a 3-
to >10-fold decreased affinity for dsDNA. Mre11-L61K and
Mre11-L97D retain high levels of ssDNA endonuclease activity
(Figure 3G) and 30-50dsDNA exonuclease activity (data not
shown), suggesting that a high-affinity DNA interaction across
the dimeric interface is not essential for efficient exo- and endo-
nucleolytic catalysis. These results dissect Mre11 catalytic and
architectural functions. Mre11 dimerization is not critical for ca-
talysis but may enable architectural DNA-binding functions of
MRN by promoting high-affinity DNA interactions. Specifically,
our SAXS and X-ray crystal structures suggest that the Mre11
dimer characterized here is the core of the Mre11 complex
DNA-binding head that recognizes diverse DNA structures and
promotes short-range DNA synapsis.
Mre11 30-50Exonuclease Mechanism
To test the structural implications of the DNA complexes for the
Mre11 exonuclease polarity and dissect determinants of exo-
and endonucleolytic catalysis, we examined the trajectories of
the 30ends of the bound dsDNAs. In both Mre11-DNA com-
plexes, the 30termini trace a path along the DNA-binding cleft
towardthe activesite,consistent withreported 30-50dsDNA exo-
nuclease directionality and the orientation of 50-dAMP nucleo-
lytic product binding (Figures 1E and 1F) (Hopfner et al., 2001;
Paull and Gellert, 1998). These structures support a three-step
30-50nuclease mechanism (Figure 4A): (1) DNA binding, (2)
duplex melting linked to capping domain rotation, and (3) phos-
phate rotation and nucleolysis driven by conserved nuclease
motif II (His52) and motif III (His85) histidines.
In the two complexes, DNA duplexes occupy similar but
nonequivalent positions and registers in the binding cleft
(Figure 4C). Structural overlays indicate DNA translocation is
linked to capping domain rotation, reorientation of the Mre11 di-
merization axis, and movement of the RL1 minor groove wedge
rotation and associated plastic deformations may drive duplex
end melting. A relay of van der Waals contacts initiated by the
capping domain and involving residues Phe16, Lys18, His17,
Tyr301,andTyr325advancestheRL1wedge intothe DNAminor
groove (Figure4C). Thenet result of thesemovements isan ?2/3
ruption of terminal base pairing interactions, as visualized for the
synaptic complex DNA, where the wedge penetrates further into
exonuclease activity, as monitored by release of the fluorescent
adenine analog 2-aminopurine from the 30end of a preferred
Mre11 blunt dsDNA exonuclease substrate (Figure 4B). Thus,
the capping domain rotation may govern the ATP hydrolysis-
lert,1999).Interestingly, P.furiosusMre11is?12-foldless active
on a branchedsubstrate (Figure S4). Stable 30end binding by the
capping domain with limited exonucleolysis or endonucleolysis
may ensure maintenance and control of the integrity of recombi-
nogenic DNA 30termini.
Base pair melting and minor groove widening helps accom-
modate DNA backbone access requirement for exonucleolytic
activity on preferred blunt or 30recessed dsDNA substrates.
Based upon the positions of the DNA 30termini in the Mn2+
bound and unbound structures (Figure 4D, gray 30terminus),
the 30end trajectory (Figure 4C, yellow 30terminus) is evident
from bound 50-dAMP in the exonuclease product structure
(Hopfner et al., 2001). After duplex melting, phosphate rotation
3 base pairs 50to the cleavage site would redirect the 30terminus
into the active-site cleft for cleavage. Two stringently conserved
histidines (motif II His52 and motif III His85) gate the 30end path
to the product-binding site (Figure 4D). Except for His52 and
His85, most residues from nuclease motifs I–V act directly in
(E) ssDNA endonuclease activity for wild-type Mre11 and nuclease mechanism variants.
(F) Alternative ssDNA-binding site identified by docking. All top DOT docking solutions for 3-mer ssDNA cluster to a groove at the capping to nuclease interface
near the active site Mn2+(pink).
(G) Minimal requirements for Mre11 30-50dsDNA exonuclease activity. Exonucleolytic catalysis on dsDNA substrates requires phosphate rotation via motif II
histidine to liberate the 30terminus from duplex DNA, Mn-dependant alignment of the scissile phosphodiester bond, and stabilization of the transition state
by motif III histidine.
phosphate rotation via the motif II histidine. Mn-dependant alignment of the substrate and transition state stabilization is critical for endonucleolytic cleavage of
Cell 135, 97–109, October 3, 2008 ª2008 Elsevier Inc. 103
metal ion binding and active-site assembly. His52 and His85 are
ternary complex structure with bound Mn2+and branched DNA
substrate shows that His85 mutation does not influence Mn2oc-
cupancy (Figure S5).
Conserved Mre11 Motifs Regulating Structure-Specific
To test our catalytic model, we examined His52 and His85 roles
in 30-50exonucleolytic and ssDNA endonucleolytic cleavage.
H52S and H85S mutants lack 30-50
(Figure 4B). His85 binds the 50-dAMP phosphate with an implied
rolein stabilizingthepentacovalent transitionstateduringhydro-
lysis (Hopfner et al., 2001), but His52 was proposed to act in
a charge relay with His85 during transition state stabilization
(Hopfner et al., 2001). Here, His52 interactions with the phos-
phate backbone suggest that this motif II histidine acts in phos-
phate rotation (Figure 4D and Figure S5). So, our data implicate
distinct critical roles for His52 and His85, with motif II His52
driving phosphate rotation for 30-50exonucleolytic activity.
DNAdocking analysissuggests thatssDNA may approach the
active site by an alternative binding mode (Figure 4F). Also, be-
cause ssDNA is flexible, we hypothesized that ssDNA endonu-
tation function to access the DNA backbone but should require
di-Mn ion-directed alignment of the scissile phosphodiester
bond and transition state stabilization by motif III His85. As pre-
dicted, the H85S mutant lacks endonuclease activity, whereas
H52S displays only slightly diminished endonucleolytic cleavage
of circular fX174 ssDNA (Figure 4E). These results suggest that
dsDNA exonuclease and ssDNA endonuclease activities have
distinct, structure-specific, DNA-alignment requirements for nu-
cleolytic cleavage (Figures 4G and 4H). Motif II mutation H52S
ablates 30-50exonuclease activity but retains endonuclease
activity. In contrast, disruption of motif III histidine His85 blocks
both Mre11 endonuclease and 30-50exonuclease activities.
DSB Repair Functions Require Mre11 Dimerization
and Endonucleolytic Activities
Guided by the P. furiosus Mre11-DNA complex structures, we
made mutant alleles of S. pombe mre11 (rad32) to test whether
Mre11 dimerization and nucleolytic activities are required for
DSB repair. We analyzed two classes of mutations: (1) dimeriza-
tion mutations deficient in DNA binding that retain exonuclease
and endonuclease activities and (2) catalytically deficient
mutants that segregate exonucleolytic and endonucleolytic
activities but do not alter metal coordination or Mre11 complex
To probe the Mre11-Mre11 interface, we substituted charged
residues for Leu77 and Leu154 in S. pombe Mre11 (P. furiosus
Mre11 Leu61 and Leu97; Figure S1). Two-hybrid analysis shows
that wild-type Mre11 had a robust self-interaction, but mutations
of dimer interface leucines severely diminished interactions
(Figure 5A, upper panels). Importantly, these mutations did not
impair interactions with Rad50 and Nbs1 (Figure 5C, upper
blocked the Mre11 self-interaction, the interface might be re-en-
gineered to associate via a salt bridging network that occupied
a similar volume (Figure 5B). This charge-restored interface de-
sign tests the need for the specific interface versus another
direct connection. Replacement of the LL/LL hydrophobic inter-
face with a KD/KD or DK/DK salt-bridging network partially
restored the interaction, as measured by two-hybrid assay (Fig-
ure 5A, lower panels). However, although the monomeric single
variantsL77K and L154Dinteract with Nbs1and Rad50, the salt-
bridge L77K/L154D dimer is defective in these interactions
(Figure 5C). Thus, precise Mre11 dimer assembly, consistent
with DNA-binding and end-pairing geometries observed in our
Mre11-DNA complex crystal structures, is critical for a functional
ins, we examined responses to four damaging agents: (1) ioniz-
ing radiation (IR), which directly makes DSBs, (2) ultraviolet (UV)
light, which creates DNA photoproducts that can be processed
into DSBs, (3) camptothecin (CPT), a topoisomerase inhibitor
that causes replication fork breakage when the replisome en-
counters a topoisomerase-CPT complex, and (4) hydroxyurea
ductase, an enzyme required for dNTP synthesis. The mre11 al-
leles replace genomic mre11+and encode a C-terminal myc tag.
These strains were compared to mre11D and to a myc-tagged
mre11+strain, which appeared to be identical to untagged
mre11+in survival assays (Figures 5D and 5E). Immunoblotting
showed that the L77K and L154D mutants express protein at
levels comparable to the wild-type, whereas L77K/L154D abun-
dance was reduced ?50%.
in yeast two-hybrid assays, the L77K/L154D double mutant re-
sembled mre11D in being very sensitive to genotoxins (Figures
5D and 5E). The L154D mutant was sensitive to the higher
dose (2 mM) of CPT but was otherwise largely genotoxin insen-
sitive. Yet, L77K was sensitive to all of the genotoxins and most
notably to CPT, but less so than was mre11D. These data show
that the dimer interface is important for Mre11 DSB repair func-
tion. The mild defect for L154D and intermediate defect of the
L77K mutant suggest that the mutations do not fully disrupt
Mre11 dimerization in vivo or that ablation of dimerization does
not fully inactivate Mre11. Although S. pombe Mre11 dimer mu-
subunit binding to dimeric Rad50, the resulting increased local
concentration of Mre11 may enable association of reduced-
affinity dimers, as seen for the P. furiosus Mre11 dimer mutants.
Intriguingly, Mre11 ATLD missense variants (hMre11 N117S and
W210C) also disrupt Nbs1 interactions (Fernet et al., 2005; Lee
et al., 2003; Stewart et al., 1999) and map to one surface on
Thus, Mre11 ATLD mutations and dimer mutants resulting in
subunit misalignment may both impact protein-protein interac-
tions by distorting the Mre11 surface, and are analogous to can-
cer-causing XPD mutations distorting domain interfaces (Fan
et al., 2008) and BRCA1 mutants that ablate protein-phospho-
peptide binding surfaces (Williams et al., 2004).
To test roles of the exonucleolytic and endonucleolytic activi-
ties in fission yeast, we employed the phosphate rotation motif II
mutation (H68S, corresponding to P. furiosus mre11-H52S) and
the nuclease transition state stabilization motif III mutation
104 Cell 135, 97–109, October 3, 2008 ª2008 Elsevier Inc.
Figure 5. Dimerization and Nuclease Activities Are Required for Mre11 Complex DNA Repair Functions in S. pombe
(A) Mre11 self-interaction by two-hybrid assays. Growth on DEX-LWHA indicates a positive two-hybrid interaction.
(B) Energy minimized model for a designed salt-bridged, reconstituted Mre11-Mre11 dimeric interface.
(C) Mre11 variant interactions with Rad50 and Nbs1 by two-hybrid analyses. Growth on Dex-LWHA indicates a positive two-hybrid interaction.
(D) Mre11 dimerization and endonuclease disruption variants are IR sensitive.
in clastogen sensitivity. Five-fold serial dilutions of cells on YES plates were photographed after 2–3 days growth at 30?C. Expression levels of myc-tagged wild-
type and mutant Mre11 proteins were determined by immunoblotting.
(F) ATLD missense mutations W210C and N117S, which impair Nbs1 binding, cluster to a single Mre11 dimer surface opposite the DNA-binding cleft.
(G) Endonuclease activity is required for efficient DSB repair in fission yeast. Mre11 catalytic activity for mutantP. furiosus proteins is compared to genotoxin resis-
Cell 135, 97–109, October 3, 2008 ª2008 Elsevier Inc. 105
(H134S, corresponding to P. furiosus mre11-H85S). Both mu-
tants displayed robust two-hybrid self-interactions, indicating
that they do not impair Mre11 dimerization (Figure 5A). Likewise,
both mutants strongly interacted with Rad50 and Nbs1 (Fig-
ure 5C). Immunoblotting showed that they were present at levels
similar to wild-type Mre11 in S. pombe (Figure 5E). Remarkably,
the H68S mutant, which is expected to be severely deficient in
30-50exonuclease activity while maintaining endonuclease activ-
ities, showed only mild CPT and IR sensitivity at high doses. In
contrast, the H134S mutant, which is expected to lack all nucle-
ase activities, showed genotoxin sensitivity approaching that of
mre11D (Figures 5D and 5E). Because Mre11 is required for HR
but not NHEJ repair in fission yeast (Manolis et al., 2001), our re-
sults suggest that the endonuclease activity of Mre11 is crucial
for HR repair of DSBs (Figure 5G).
Exo1 Rescues mre11-H134S
Because the nuclease-deficient mre11-H134S is highly sensitive
to genotoxins, we examined whether these phenotypes show
defects in processing DNA ends and whether they may be linked
to Ctp1, a critical cofactor for MRN 50to 30resection function
(Limbo et al., 2007). Mre11 and Ctp1 deficiency are partially sup-
pressed by elimination of the Ku80 subunit of the Ku70-Ku80
DNA end-binding complex that promotes NHEJ and protects
(Limbo et al., 2007; Tomita et al., 2003). A pku80D mutation
suppressed the slow-growth phenotype and IR, CPT, and UV
sensitivities of mre11-H134S cells (Figure 6A). Importantly, the
extreme genotoxin sensitivity of the mre11-H134S pku80D
exo1D strain showed that this suppression was dependent on
Exo1 activity. Indeed, the exo1D mutation substantially exacer-
bated the mre11-H134S phenotypes. Similar genetic interac-
tions are seen in S. cerevisiae, although the double-mutant phe-
notype is mild compared to that of mre11D (Moreau et al., 2001).
The ability of Exo1 to suppress mre11-H134S strongly suggests
properly resect DSBs.
Because Ctp1 is required for the MRN-dependent processing
of DSBs and Mre11 is required for localization of Ctp1 at DSBs
(Limbo et al., 2007), it was important to establish whether the
mre11-H134S phenotype might be a Ctp1 recruitment defect.
We therefore used a chromatin immunoprecipitation (ChIP) ex-
periment to determine whether mre11-H134S cells recruit Ctp1
to a DSB created by HO endonuclease. This analysis detected
robust enrichment of Mre11-H134S, Nbs1, and Ctp1 to a region
0.2 kb from the DSB (Figures 6B and 6C). As seen before (Limbo
et al., 2007), phosphorylation of histone H2A (g-H2A) was
detected at regions further from the DSB (2–16 kb) but not 0.2 kb
from the DSB site. These data show that the H134S mutation
does not interfere with the Mre11 association with DSBs,
nor does it impair recruitment of Nbs1 or Ctp1. Therefore,
mre11-H134S defects likely reflect specific impairment of
Consistent with our EM (Hopfner et al., 2001) and X-ray struc-
tures, AFM shows that heterotrimeric yeast Mre11-Rad50-Xrs2
with the globular DNA-binding head localized at the junction of
and bridge the diverse DNA architectures encountered at breaks
or ssDNA-dsDNA junctions, such as collapsed or restarting
Figure 6. Exo1 Substitutes for Mre11-H134S in DSB Repair
(A) The CPT and IR survival defects of mre11-H134S are suppressed by elimination of Ku80, and the rescue depends on Exo1.
and act1 locus, and their product sizes were 285 bp, 213 bp, 162 bp, 145 bp, and 121 bp, respectively.
(C) MRN and Ctp1 localize to aDSB in mre11-H134S. ChIP assay for localization of g-H2A, Mre11, Nbs1, and Ctp1 to a HO-inducedDSB is shown for the mre11-
H134S nuclease-deficient mutant. Samples were collected at the indicated time after HO expression. Phospho-H2A is localized at 16, 9, and 2 kb, which is
indicative of efficient HO-break formation. Mre11, Nbs1, and Ctp1 are localized specifically at 0.2 kb upon DSB formation.
106 Cell 135, 97–109, October 3, 2008 ª2008 Elsevier Inc.
replication forks (Costanzo et al., 2001; Trenz et al., 2006; Wen
et al., 2008). These results explain and extend observations
that the Mre11 complex forms DNA damage repair nuclear foci
upon replication fork collapse in budding yeast (Lisby et al.,
2004), prevents accumulation of DSBs during replication in
Xenopus extracts (Costanzo et al., 2001; Trenz et al., 2006),
and is an early sensor of IR (Mirzoeva and Petrini, 2001) or endo-
nuclease-induced DSBs (Lisby et al., 2004). Thus, the Mre11
complex harbors adaptable and multivalent DNA-bridging capa-
bilities and utilizes distinct modes of long-range and short-range
DNA tethering to sense, coordinate, process, and port variable
DNA ends to HR repair proteins (Figure 7).
nucleaseactivities inDSBrepair.Themildphenotypes displayed
for the S. pombe phosphate rotation H68S mutant suggest that
a robust Mre11 30-50exonuclease activity is not critical for HR re-
gesting that Mre11 endonuclease activity is crucial for HR repair
of DSBs, although 30-50exonuclease activity may aid resection.
The H134S mutation is a nucleolytic defect because Mre11-
H134S maintains robust interactions with Rad50, Nbs1, and
Mn2+and also mediates Nbs1 and Ctp1 recruitment to DSBs.
The mre11-H134S mutation’s strong phenotype in fission
show mild IR sensitivity (Krogh et al., 2005; Lewis et al., 2004).
Yet, an analogous murine Mre11-H129N maintains Nbs1 and
Rad50 interactions but shows impaired recruitment of RPA and
Rad51 into ionizing radiation-induced nuclear foci and a 90% re-
duction in HR repair efficiency, consistent with our genetic data
suggesting a 50-30resection defect for S. pombe Mre11-H134S.
Moreover, Mre11-H129N also confers early embryonic lethality
in mice, and conditional knockin of this allele causes severe IR
hypersensitivity (Buis et al., 2008).
We therefore propose that Mre11 endonuclease activity acts
inconjunction with additional factors thatopenDNAendstopro-
duce an effective 50-30excision process that liberates 30ssDNA
forRPAloadingand initiationof strandinvasion inearlyHRrepair
steps in vivo. This function is likely mediated by the eukaryotic
Ctp1 (CtIP) protein, which is required for HR repair in S. pombe
and human cells and modifies the human Mre11 complex endo-
nuclease activity in vitro (Limbo et al., 2007; Sartori et al., 2007).
Ctp1 (CtIP) is a probable ortholog of budding-yeast Sae2, which
has a hairpin-nicking activity in vitro (Lengsfeld et al., 2007). In-
terestingly, budding-yeast sae2D mutants have a mild IR-sensi-
tive phenotype (Rattray et al., 2001) similar to the mre11-H125N
or H125S nuclease mutations, whereas fission yeast ctp1D mu-
tants have a more severe IR-sensitive phenotype equivalent
(Limbo et al., 2007). These differences may reflect that bud-
ding-yeast MRX has acquired additional nuclease-independent
NHEJ functions, and differing contributions of additional nucle-
ases to DSB processing (Moreau et al., 2001).
Our combined observations suggest Mre11 is crucial for
sensing, processing, and coordinating control of both ssDNA
and dsDNA during HR repair. Mre11 endonucleolytic cleavage
of ssDNA and regions neighboring transiently formed second-
ary structures contributes to the 50-30resection processing
reaction (Figure S6) (Trujillo and Sung, 2001). In contrast, the
Figure 7. Mre11 Dimer Holds Together and Processes Both Two-
Ended Breaks from Damage and One-Ended Breaks Arising during
The MRN complex coordinates processing of DSBs arising from exogenous
damage (left panels) or during replication (right panels).
(A) Two-ended DSBs can form with exposure to IR, whereas blocks to DNA
replication can create one-ended DSBs.
face mutations. Mre11 complex scaffolding facilitates and coordinates DSB re-
Mre11 dimer-mediated DNA synapsis and by Rad50 hook-dependant long-
tionsthroughthe Rad50hook can tetherthe brokenDNA endstosisterchroma-
tids, thereby facilitating recombination repair. Single-end binding and branched
on one-sided double-strand breaks formed from collapse of replication forks.
(C) Mre11 endonuclease activity in the context of DNA unwinding is required
for 50-30resection to generate 30tails for strand invasion. Mre11 nuclease
mutation H134S disrupts DSB processing steps for initiation of HR repair.
(D) Homologous recombination repair initiated by liberated 30termini.
Cell 135, 97–109, October 3, 2008 ª2008 Elsevier Inc. 107
Mre11 30-50exonuclease is well suited for Mre11 complex roles
in 50-microhomology-mediated end-joining pathways (Fig-
ure S7) (Paull and Gellert, 1998, 2000). Employment of two jux-
taposed DNA-binding clefts may help validate repair status by
sensing an empty DNA-binding cleft, versus one or two bound
DNA ends. Furthermore, as seen for the FEN-1 complex with
dsDNA (Chapados et al., 2004), controlling the 30primer termi-
nus at a strand break may be a critical function for the Mre11
complex. Together, these results highlight the Mre11 structural
and enzymatic contributions within the DSB repair complex to
the biological needs for both creating and controlling proper
30-OH ends to prime DNA repair synthesis.
Protein Expression and Purification
Expression and purification of wild-type and mutant P. furiosus Mre11 (resi-
dues 1–342) followed published procedures (Hopfner et al., 2001). The
H52S,H52S, L77K, and L97D mutants were introducedby Quickchange (Stra-
Crystallization, X-Ray Diffraction Data Collection, Structure
Determination, and Refinement
We employed four-thymine hairpin DNA architectures to promote directional
binding of duplex ends during crystallization (Figure S8).For thesynapticcom-
plex, Mre11 (at 30 mg/ml in 200 mM NaCl, 20 mM TrisHCl [pH 7.5], 1 mM di-
thiothreitol [DTT], and 0.5 mM ethylenediaminetetraacetic acid [EDTA]) and
DNA substrate (50-CAC AAG CTT TTG CTT GTG AC-30) were mixed at a 3:1
DNA:protein molar ratio. Complex crystals (space group C222) were grown
with sitting-drop vapor diffusion at 20?C–22?C by mixing of 1 ml Mre11-DNA
complex with 1 ml reservoir solution 1 (20% polyethylene glycol 1000, 0.1 M
Tris-HCl [pH 7.5], 0.2 M magnesium chloride, and 0.2 M 1,6 hexanediol).
Data to 2.7 A˚were collected at 105?K from a crystal transferred to cryoprotec-
tant solution 1 (20% [v/v] ethylene glycol, 16% PEG1000, 0.08 M Tris-Cl
[pH 7.5], 0.16 M magnesium chloride, and 0.16 M 1,6 hexanediol).
For the Mre11-His85Ser/Mn2+/Branched-DNA ternary complex, Mre11-
H85S (at 10 mg/mL in 100 mM NaCl and 20mM Tris HCl [pH 7.5]) was mixed
at a 3:1 DNA:protein ratio with the branched oligonucleotide hairpin (50-CGC
GCA CAA GCT TTT GCT TGT GGA TA-30) 30 min before crystallization. By
hanging-drop vapor diffusion at 22?C, orthorhombic crystals (space group
P212121) grew within 1 week after mixing of 1 ml complex with 1 ml reservoir
solution 2 (40% v/v Polyethylene glycol 200 and 100 mM HEPES [pH 7.5]).
Crystals were flash cooled in liquid nitrogen directly from hanging drops,
and diffraction data was collected to 2.2 A˚(see Table S1).
Dataprocessing,phasing,model building, and refinementaredetailed inthe
Supplemental Data. Final refined models of the synaptic complex at 2.7 A˚(R/
Rfree= 22.8/27.8) and branched complex at 2.2 A˚(R/Rfree= 20.0/24.6) exhibit
good geometric parameters (Table S1).
Small-Angle X-Ray Scattering
SAXS data for wild-type PfMre11 catalytic domain and dimerization mutants
(PfMre11-L61K, L97D) were acquired at the Advanced Light Source SIBYLS
beamline (BL12.3.1). Fifteen microliter samples in a 1 mm thick sample cell
equipped with 25 uM Mica windows at a sample to detector distance of
1.5 m were illuminated at 10 keV (l = 1.1159 A˚) for data collection (q range =
0.010 A˚?1–0.250 A˚?1). Scattering profiles in SAXS buffer (500 mM NaCl,
20mM Tris [pH 7.5], 5% glycerol, and 1 mM DTT) were collected from
10 mg/mL samples and analyzed (see the Supplemental Data).
Nuclease and Electrophoretic Mobility Shift Assays
Mre11 exonuclease and endonuclease assays were performed as described
(Hopfner et al., 2001) with substrates detailed in the Supplemental Data.
DNA-binding reactions for EMSAs were carried out in 25 mM HEPES
(pH 8.0), 50 mM NaCl, 1 mM DTT, 0.1 mg/ml acetylated bovine serum albumin,
and 10% glycerol containing 50 fmol of DNA substrates (see the Supplemental
Strain Construction and Plate Survival, Yeast Two-Hybrid,
and HO ChIP Assays
Plate and IR survival assays were performed as described (Limbo et al., 2007)
for strains described in the Supplemental Data. Two-hybrid assays were
performed as previously described (Limbo et al., 2007). Chromatin immuno-
precipitation assays were conducted as described previously (Limbo et al.,
Whole-cell extracts were prepared from exponentially growing cells. Cells
were lysed in 0.5% b-mercaptoethanol and 0.3M NaOH, and protein was pre-
cipitated in 25% trichloroacetic acid, resuspended in SDS-PAGE sample
buffer, and incubated at 65?C for 15 min. Western blotting was performed
with anti-Myc (9E10; Santa Cruz Biotechnology) and pstair (Sigma) antibodies.
Structures of the Mre11-synaptic DNA complex (PDB ID code 3DSC) and
Mre11-branched DNA complex (PDB ID code 3DSD) are in the Protein Data
Supplemental Data include Supplemental Experimental Procedures, eight fig-
ures, and four tables and can be found with this article online at http://www.
R.S.W.issupportedby theCanadian Institutes ofHealth Research, theAlberta
HeritageFoundationforMedicalResearch,and theSkaggs InstituteforChem-
ical Biology fellowships. Y.Y. is supported by a Uehara Memorial Foundation
fellowship. Work on the Mre11 complex in the authors’ laboratories is sup-
ported in part by National Cancer Institute grants CA117638, CA92584, and
CA77325. Lawrence Berkeley National Laboratory efforts are supported in
part by U.S. Department of Energy programs IDAT (for Integrating crystallog-
raphy and X-ray scattering) and MAGGIE (for defining Pyrococus complexes)
under contract number DE-AC02-05CH11231. We thank Scott Classen, Mi-
chal Hammel, Greg Hura, and Susan Tsutakawa for assistance with SAXS
and X-ray data collection at the Advanced Light Source SIBYLS beamline
and Brian Chapados for discussions and comments.
Received: March 4, 2008
Revised: June 17, 2008
Accepted: August 7, 2008
Published: October 2, 2008
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