Available online at www.sciencedirect.com

Structural biology of DNA photolyases and cryptochromesMarkus Muller and Thomas Carell

Photolyases repair cytotoxic and mutagenic UV-induced

photolesions in DNA by using an amazing light-dependent

repair mechanism. It involves light absorption, electron transfer

from an excited reduced and deprotonated FADH� to the

flipped-out photolesion, followed by the fragmentation of the

photolesions. Cryptochromes are highly related proteins that

no longer repair damaged DNA, but function as

photoreceptors. They feature strikingly similar protein

architectures to photolyases and contain an FAD cofactor as

well. However, cryptochromes possess an additional signal-

transmitting domain, attached either to the N-termini or C-

termini. Recently, the field of photorepair and blue-light

photoperception has experienced significant progress

particularly in structural biology, which is summarized in this

review. Today, crystal structures of many family members are

known and most recently even complexes of photolyases and

DASH-type cryptochrome bound to their DNA substrates

became available providing insight into the critical electron and

energy transfer reactions that enable genome repair.

AddressCenter for Integrated Protein Science CiPSM at the Department of

Chemistry and Biochemistry, Ludwig-Maximilians University Munich,

Butenandtstr. 5-13, D-81377 Munich, Germany

Corresponding author:

Carell, Thomas (thomas.carell@cup.uni-muenchen.de)

Current Opinion in Structural Biology 2009, 19:277–285

This review comes from a themed issue on

Nucleic acids

Edited by Eric Westhof and Dinshaw J Patel

Available online 30th May 2009

0959-440X/$ – see front matter

Published by Elsevier Ltd.

DOI 10.1016/j.sbi.2009.05.003

IntroductionEarly research of DNA repair started with the observation

that bacteria treated with UV-light showed a higher

survival rate after a second irradiation step with blue

light. This enigmatic photoreactivation process was soon

correlated with the function of DNA photolyases [1],

which utilize blue light to clean the bacterial genome

from UV-induced lethal lesions. The field of the photo-

lyase/cryptochrome family has been summarized in a

number of excellent reviews [2��,3]. Here we cover the

most recent advances in the structural biology of photo-

lyases and cryptochromes. Today, DNA photolyases are

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considered to be ancient DNA repair proteins, which

helped already the first living organisms exposed to sun-

light to survive. Upon using a light-induced repair reac-

tion, photolyases revert cyclobutane–pyrimidine-dimers

(CPD) and so-called (6-4) pyrimidine–pyrimidone photo-

lesions inside DNA back into undamaged pyrimidine–pyrimidine dinucleotides. As such they are essential for

genome maintenance in many species. From these photo-

lyases a special class of blue-light photoreceptors evolved,

the so-called cryptochromes. These proteins have lost

their ability to repair photolesions, but act in photoper-

ception and signal transduction of blue light in a broad

range of organisms. In plants, cryptochromes control for

example hypocotyl growth and the transition to flowering

[4]. In mammals, they are central components of the

circadian clock [5]. In birds, cryptochromes are even

supposed to be component of the magnetoperception

system [6,7]. The main difference between photolyases

and cryptochromes is the presence of C-terminal or N-

terminal extensions in cryptochromes that mediate the

signal transduction step. Until today, however, these

extension domains remained inaccessible to structural

analysis so that any information about their mechanism

of function is lacking. Only the short N-terminal exten-

sion of Arabidopsis Cry3 (AtCry3) was structurally charac-

terized, but its function in signal transduction is yet

unknown. However, crystallographic data are available

for the photolyase-like domain of AtCry1.

Evolution of the photolyase/cryptochromefamilyDespite the presence of photolyase and cryptochrome

genes in organisms belonging to all three kingdoms of life

(archaea, bacteria, and eukarya), the evolutionary con-

nections (Figure 1) of the individual members of this

unique protein family are still discussed controversially.

Today, it is widely accepted that the common ancestor

was likely a CPD-photolyase. This protein subdivided

into the today known class I (yellow in Figure 1), class II,

and the recently identified class III subfamilies (not

shown), of which the plant cryptochromes evolved

(green) [8]. The (6-4) photolyases (orange) and the closely

related animal cryptochromes (purple) represent another

distinct branch. In 2003 Brudler et al. [9�] described a new

class of cryptochromes named cryptochrome-DASH

proteins (DASH = Drosophila, Arabidopsis, Synechocys-

tis, Homo, shown in blue). On the basis of sequence

alignment, they grouped these proteins together with

the animal cryptochromes and (6-4) photolyases. How-

ever, Kleine et al. [10] proposed that the plant Cry-DASH

protein named AtCry3 originated from ancient cyanobac-

teria (chloroplasts) while AtCry1/2 were derived from

Current Opinion in Structural Biology 2009, 19:277–285

278 Nucleic acids

Figure 1

Phylogenetic tree of representative members of the cryptochrome/photolyase family, calculated with ClustalX [47]. Organisms with structures available

are listed at their respective position in the tree. The class II and III photolyases were omitted from the tree to provide better clarity. Surrounding:

substrate-bound structures of photolyase/cryptochrome proteins 1TEZ, 2VTB, 3CVU, and 1U3D. The N-terminal extension of AtCry3 is colored in

orange.

a-proteobacteria (mitochondrial). Recent data show now

that the DASH cryptochromes are single-strand-specific

CPD-photolyases [11,12�], which currently shift our per-

ception of these proteins.

Common structural featuresToday, several crystal structures of photolyase and cryp-

tochrome proteins are available. The first crystal struc-

tures obtained in 1995 [13��] and 1997 [14] showed the

CPD-photolyases from Escherichia coli and Anacystis nidu-lans. Both structures revealed the three-dimensional

architecture of these DNA repair proteins in the absence

of the respective DNA substrates. This left the question

of how the electron transfer occurs initially open. How-

ever, the structures showed that the proteins possess a

rather globular structure, uncommon to most DNA bind-

ing proteins. Both structures display the same domain

architecture with an N-terminal a/b domain and a C-

terminal a-helical domain. These photolyases contained

two cofactors. The a-helical domain is harboring the

flavin-adenine-dinucleotide (FAD) cofactor, essential

Current Opinion in Structural Biology 2009, 19:277–285

for catalysis. The second cofactor, which is either an 8-

hydroxy-5-deazariboflavin (8-HDF) or 5,10-methenylte-

trahydrofolate (MTHF), is always bound at the interface

between these two domains, about 9 A away from the

FAD as shown in Figure 2b.

In 2004 the first cocrystal structure of a CPD-photolyase

from A. nidulans in complex with a DNA-duplex contain-

ing a synthetic CPD lesion analog was reported [15��],showing that the protein fold changed only marginally

upon DNA binding. The structure proved that CPD-

photolyase proteins fully open the DNA-duplex structure

at the damaged site and flip the dinucleotide lesion out of

the duplex into the active site. Here the lesion comes in

close contact (3 A) with the active FADH� cofactor. Until

today, photolyases are the only proteins, which are able to

perform such a dramatic dinucleotide flip-out.

The first crystal structures of the photolyase-like domains

of bacterial and plant cryptochromes [9,16,17,18�],reported between 2003 and 2007, revealed a surprising

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DNA photolyases and cryptochromes Muller and Carell 279

Figure 2

(a) Schematic representation of the photoreactivation process. Oxidized FAD is reduced to FADH� in a light-dependent process called photoactivation

that involves electron transfer toward FAD from conserved tryptophane and tyrosine residues. Absorbed light energy is transferred from the accessory

pigments to FADH� by Forster-energy-transfer (FRET), enabling electron transfer and splitting of the CPD-lesion or (6-4)-lesion. (b) Superimposed

electron-transfer and energy-transfer paths in Anacystis nidulans CPD-photolyase (green) and Drosophila melanogaster (6-4) photolyase (red, from

[40]). The numbering is according to Anacystis PHR.

homologous fold topology between all these proteins.

The basic folds of these domains and of CPD-photolyases

are about the same. Most recently, the first crystal struc-

ture of a (6-4) photolyase bound to a DNA-duplex con-

taining a synthetic (6-4) lesion substrate [19�] was

obtained and again the observed protein architecture

was found to be the same. This observation now extends

even to the structure of the Arabidopsis cryptochrome 3 in

complex with a single-stranded DNA strand containing

the same synthetic CPD lesion analog [11]. The structure

of an animal cryptochrome remains yet to be solved but

one can suspect that the structure will be similar as well.

Despite a high level of sequence diversity (41.9% sim-

ilarity, 5.6% identities), the topological similarity be-

tween all photolyase and cryptochrome structures

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known today is one of the most surprising discoveries.

The total rmsd (root mean square deviation) for 9 struc-

tures with varying sequences (Table 1) is only 1.914 A

over 369 residues as calculated with SSM (http://www.e-

bi.ac.uk/msd-srv/ssm) [20]. It seems that all these proteins

utilize similar energy and electron transfer steps which,

owing to the sensitivity of these processes to distance and

orientation changes, force the proteins to keep a common

fold in order to ensure an optimal arrangement of the

essential cofactors [21,22] (Figure 2b).

For photolyases it is known that they utilize the FAD

cofactor in the reduced and deprotonated form (FADH�)

as an electron donating entity (Figure 2a). It is today

believed that the reduction of FAD to FADH� is the first

Current Opinion in Structural Biology 2009, 19:277–285

280 Nucleic acids

Table 1

A list of all to this date available photolyase and cryptochrome structures

Protein Organism PDB-ID Secondary cofactor Substrate Reference

CPD-Phr Escherichia coli 1DNP MTHF None [13��]

CPD-Phr Anacystis nidulans 1QNF 8-HDF None [14]

1TEZ 8-HDF T< >T [15��]

1OWL, 1OWM, 1OWN, 1OWO, 1OWP None None [45]

CPD-Phr Thermus thermophilus 1IQR, 1IQU None Thymine [46]

2J07, 2J08, 2J09 FMN, 8-HDF, 8-IRF None [38]

CPD-Phr Sulfolobus tokodaii 2E0I FAD None [39]

(6-4)-Phr Drosophila melanogaster 3CVU, 3CVY None T(6-4)T [19�]

3CVV, 3CVW, 3CVX 8-HDF, none T(6-4)T [40]

(6-4)-Phr Arabidopsis thaliana 3FY4 None None [37]

Cry1 Arabidopsis thaliana 1U3C, 1U3D None AMP-PNP [18�]

Cry-DASH Synechocystis spp. 1NP7 None None [9�]

Cry3 Arabidopsis thaliana 2IJG MTHF None [17]

2J4D MTHF None [16]

2VTB MTHF T< >T [11]

The rmsd of the nine structures with varying sequences is 1.914 A (1DNP, 1QNF, 1IQR, 2E0I, 3CVU, 3FY4, 1U3C, 1NP4, and 2IJG) as calculated with

SSM [20]. Phr, photolyase (photorepair); MTHF, 5,10-methenyltetrahydrofolate; 8-HDF, 8-hydroxy-5-deazariboflavin; 8-IRF, 8-iodo-8-demethyl-

riboflavin; FMN, flavin-mononucleotide; FAD, flavin-adenine-dinucleotide; T< >T, cyclobutane–pyrimidine-dimer-containing DNA; T(6-4)T, (6-4)

pyrimidine–pyrimidone-dimer containing DNA; AMP-PNP, adenosine 50-(b,g-imido)triphosphate.

required photochemical step (photoactivation). Initial

photoexcitation of FAD is supposed to trigger sequential

electron donations from three conserved tryptophanes

[23] and an additional tyrosine residue in Anacystis photo-

lyase [24], followed by a proton uptake. The tryptophane

cascade needed for FAD reduction is present in all

available photolyase/cryptochrome structures, listed in

Table 1. Next, the repair process involves absorption

of light by the MTHF/8-HDF antenna chromophores,

followed by energy transfer from the excited antenna

pigments to the FADH� and finally electron transfer to

the CPD or (6-4) lesion. The lesion then splits back into a

dipyrimidine dinucleotide as depicted in Figure 2a. The

electron-transport chain of AtCry1, like in Anacystis photo-

lyase, involves a Tyr residue as initial electron donor.

However, the active signaling state of the cryptochromes

was shown to be the semireduced FADH� [25]. Very

recently, comparative redox titrations of photolyase and

Cry1 have shown remarkable differences in the FAD

redox potential, favoring light-induced transitions be-

tween FADox and FADH� in Cry1 as compared to

FADH� to FADH� transitions in photolyase [26]. These

differences were attributed to an asparagine residue (Asn-

386) in the active site of E. coli photolyase, which is

replaced by Asp-396 in Cry1.

DNA bindingAlthough efficient lesion specific but sequence-indepen-

dent DNA binding was early on established for CPD and

(6-4) photolyases, the DNA binding characteristics of

cryptochromes remain to be enigmatic. Photolyases

invade the duplex and flip the lesion out of the duplex

into a rather hydrophobic lesion binding pocket. This

pocket features aromatic Trp residues, which pack

against the lesion holding it in the flipped-out state by

using p–p interactions. A large set of H-bonds established

Current Opinion in Structural Biology 2009, 19:277–285

to the Watson–Crick H-bond donors and acceptors of the

lesions provide additional binding energy to stabilize the

energetically highly unfavored flipped-out state. The gap

created in the duplex after the dinucleotide flip-out is

stabilized by amino acids G397-F406 in Anacystis CPD-

photolyase, which invade the duplex.

The fact that the cryptochrome proteins originate from

DNA repair proteins fueled the idea that they might bind

DNA as well, for example as light-triggered transcription

factors. For Cry-DASH and Arabidopsis Cry3, weak but

unspecific DNA binding was indeed observed [9�,10].

However, for Arabidopsis Cry1, DNA binding could not

be detected. Human Cry2 protein was shown binding to

ssDNA with high affinity (KD �5 � 10�9). Binding to

ssDNA containing a (6-4) photolesion is even better [27�].Yet, the protein is unable to repair the (6-4) lesions, which

is surprising, taking into account that (6-4) photolyases

and mammalian cryptochromes are closely related.

Photolyases exhibit a positively charged surface near the

substrate-binding pocket which promotes interactions

with DNA (Figure 3). All photolyases share these posi-

tively charged DNA binding surfaces. Cry1 from Arabi-dopsis thaliana, which does not bind to DNA, possesses a

strongly reduced surface charge around the FAD binding

pocket. However, Arabidopsis Cry3 features an even

more pronounced positive charge density at the putative

DNA binding site compared to the CPD-photolyase from

E. coli, but it binds DNA only weakly [10]. In addition,

AtCry2, whose modeled surface largely resembles that of

AtCry1, does bind to DNA (Alfred Batschauer, unpub-

lished results [49]). DNA binding was also firmly estab-

lished for AtCry3. Figure 3 shows surface charges

calculated with APBS [28] for several members of the

photolyase/cryptochrome family. The structures of

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DNA photolyases and cryptochromes Muller and Carell 281

Figure 3

Surface-charge representation of various photolyase/cryptochrome proteins calculated with APBS and visualized with PyMOL. All views face the FAD-

binding cavity. The following data-files were used: AtCry1 — 1U3D, AtCry3 — 2VTB, AnCPD-PHR — 1TEZ, Dm(6-4)-PHR — 3CVU. AtCry2 and hCRY1

were modeled in First Approach Mode on the SWISS-MODEL server [29] from 1U3C and 3CVU, respectively.

Arabidopsis Cry2 and human Cry1 were generated in silicoon the Swiss Modeling Server [29] in First Approach modefrom Arabidopsis Cry1 and Drosophila (6-4) photolyase

structures, respectively (rmsd 0.23 A and 0.31 A). In

summary it is now clear that the surface charge alone

is not a sufficient determinant for DNA binding. Huang

et al. [17] proposed that the (di)nucleotide flip-out is the

key step in DNA recognition. They evaluated the

physical properties of residues comprising the putative

substrate binding cavity. While photolyases share a con-

served hydrophobic cavity able to accommodate the

lesions, this cavity contains more charged residues in

Cry3. The authors argue that these charges destabilize

the putative substrate in the ‘flipped-out’ conformation

thus preventing efficient dsDNA binding. These results

were confirmed experimentally by Pokorny et al. [11],

showing that AtCry3 does repair CPDs when the lesion is

located in a preflipped out state such as in bulges of

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dsDNA. Taken together, the data suggest that positive

surface charges promote unspecific DNA binding while

tight binding requires a kind of base flip and nucleotide

binding in a properly hydrophobic/aromatic binding

pocket.

Mechanism of functionThe key element of the photolyase repair mechanism

depicted in Figure 2a is the electron transfer from the

excited FADH� to either the CPD or (6-4) lesion. This

induces in both cases a fragmentation reaction. The active

sites of all crystallized photolyases with their interacting

residues and substrates are shown in Figure 4c–e. For CPD

lesions it is known that the single electron reduced cyclo-

butane ring undergoes a thermally forbidden (Woodward–Hoffmann) [2 + 2] cycloreversion reaction followed by

back transfer of the ‘enabling electron’ to the semireduced

FADH�. In (6-4) photolyases, the fragmentation reaction is

Current Opinion in Structural Biology 2009, 19:277–285

282 Nucleic acids

Figure 4

(a) Proposed reaction mechanism of (6-4) photolyase, based on prerepair and postrepair crystal structures, taken from [19�]. (b) Superposition of

bound substrates in CPD-photolyase (1TEZ, gray) and Arabidopsis Cry1 (1U3D, green). (c) The catalytic site of Anacystis nidulans CPD-PHR, (d)

Drosophila melanogaster (6-4)-PHR and (e) Arabidopsis thaliana Cry3. Interacting residues as calculated with Ligplot [48] are shown in gray, the

photolesion in green and the catalytic FAD in pink. Hydrogen bonds are shown as dashed lines. Note that AtCry3 (e) is shown reversed as compared to

the other structures, for better visibility.

less clear, because repair requires cleavage of one C–C

bond and transfer of a hydroxyl group from the dihydro-

pyrimidine to the pyrimidone ring. Until recently it was

believed that the enzyme stabilizes an oxetane intermedi-

ate, formed after a light-independent reaction of the

C(5)OH group with the acylimine substructure of the

second ring [30]. Two active site histidines were postulated

to promote this reaction [31]. The first was thought to be

involved in deprotonating the hydroxyl group, while the

second was believed to activate the acylimine. This oxe-

tane intermediate was supposed to be cleaved by light-

induced electron injection very similar to the CPD-photo-

lyase catalyzed cycloreversion of a cyclobutane structure.

Two crystal structures of the (6-4) photolyase from Droso-phila bound to lesion containing DNA before and after

repair showed that repair does not involve oxetane for-

mation before light-induced electron transfer. The histi-

dine 369, supposed to activate the acylimine, is in a position

that does not allow efficient proton donation and hence

activation of this substructure [19�] (see Figure 4d). In

addition, Weber and collaborators showed, using ENDOR

Current Opinion in Structural Biology 2009, 19:277–285

spectroscopy, that the second histidine residue originally

believed to deprotonate the lesion’s hydroxyl group is

likely in the protonated state, functioning as a general acid

[32]. In this scenario, His-365 would be unable to depro-

tonate the C5-hydroxyl group. On the basis of these

observations we proposed recently a slightly modified

reaction mechanism, which involves direct electron

donation to the (6-4) lesion followed by cleavage of the

C–OH bond (Figure 4a). Attack of the acylimine is fol-

lowed by the activated OH group to give a radical inter-

mediate, which can rapidly fragment to give the repaired

TT dinucleotide.

In the case of the cryptochromes, the mechanism of action

remains unclear. It was clearly demonstrated that the C-

terminal extensions mediate a constitutive blue-light

response when expressed separatly from the photo-

lyase-like domain [33]. The Sancar group showed that

the mainly disordered C-terminal extension of AtCry1

interacts light-specifically with its photolyase-like domain

[34]. Further, it was demonstrated that AtCry1 undergoes

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DNA photolyases and cryptochromes Muller and Carell 283

autophosphorylation after light absorption [35] and its

crystal structure is showing a bound ATP analog at the

active site [18�]. Together, these observations make a

light-dependent conformational switching mechanism

appear plausible. However, whether or not the AMP-

PNP was bound specifically or represents a crystallo-

graphic artifact is not clear. The nucleoside triphosphate

analog is positioned differently compared to the dimer-

lesion in photolyases, with the ribose ring facing the FAD

binding pocket (Figure 4b). Nevertheless, the position

and function of substrate-contacting residues is similar. It

was hypothesized that binding of ATP blocks access to

the FAD-binding site and thus inhibits electron transfer.

AtCry3 contains all required motifs of a cryptochrome-

signal transducing domain [16,36]. However, the N-term-

inal extension in AtCry3 is positioned so far away from the

active site (Figure 1), hence providing no evidence to

support the theory of a light-dependent conformational

change. The latest addition to the family of photolyase

structures is giving rise to new ideas about the signal

transduction of mammalian cryptochromes. In the struc-

ture of Arabidopsis thaliana (6-4) photolyase, a phosphate

anion was found bound to Glu-243 and Trp-238, close to

FAD. Phosphorylation of a serine found at the same

position in the model of human Cry1 was shown to

modulate its function [37].

The light antennaDespite the overall structural similarity of photolyases

and cryptochromes, a few elements are different. Most

important are different binding-modes of the MTHF in

photolyases and Cry-DASH proteins. Between AtCry3

and E. coli CPD-photolyase, only a single glutamic acid

residue is conserved for MTHF binding [16], but still the

overall structural arrangement is very similar. The photo-

lyase from Thermus thermophilus can bind next to the

natural cofactor 8-HDF also FMN. It is unclear if this

is physiologically relevant. But seemingly, its binding

pocket is more promiscuous than those of other proteins

belonging to this family. Even the synthetic 8-iodoflavin

was found in the photoantenna pocket when crystals were

soaked with this compound [38]. In Sulfolobus tokodaii, a

second FAD molecule was also found in the antenna

pigment binding pocket [39]. The (6-4) photolyase from

Drosophila melanogaster is unexpectedly binding 8-HDF

[40]. As this cofactor was previously believed to occur only

in archaea and cyanobacteria the observation of this

signature molecule for the archaeal world in a eukaryotic

protein came as a surprise. In AtCry1 and SynechocystisCry-DASH, a secondary cofactor was not yet found.

However, biochemical data suggest that MTHF func-

tions in these proteins as the light antenna [41,42].

ConclusionsFifteen years ago CPD-photolyases were considered to be

solitary proteins, which utilize a strange light-dependent

mechanism to clean the genomes of organisms from the

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lesions induced by UV irradiation. In 1958, Claud S.Rupert discovered the first photolyase [1]. In 1993 the

first (6-4) photolyase was found [43] and in the same year

the protein family grew even further by the discovery of

cryptochromes [44]. Cryptochromes and photolyases now

form a family of proteins that absorb blue light to drive

DNA repair and control various biological processes.

Using protein crystallography it was possible to gain

insight into the structures of these proteins and to charac-

terize the DNA binding mode, which revealed for the first

time a dinucleotide flip-out process. Although the mech-

anism of action of CPD and (6-4) photolyases is now well

established, the process allowing cryptochromes to trigger

circadian rhythms and to fuel signaling cascades by blue-

light absorption remains an open question.

AcknowledgementsWe thank the Excellence Cluster CiPSM and the Sonderforschungsbereich749 for generous financial support. Many thanks to Sabine Schneider andAndreas Glas for the help with the figures and critical reading of themanuscript. All structural figures were prepared with PyMol (DeLanoScientific).

References and recommended readingPapers of particular interest, published within period of review, havebeen highlighted as:

� of special interest

�� of outstanding interest

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2.��

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This review has set the ‘standards’ of photolyase and cryptochromeresearch.

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4. Lin C: Plant blue-light receptors. Trends Plant Sci 2000,5:337-342.

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9.�

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This article presents the first structure of a cryptochrome and describes anew family of these proteins, the Cry-DASH family.

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