Báo cáo khoa học: Physiological truncation and domain organization of a novel uracil-DNA-degrading factor pdf

Sequence alignments and limited proteolysis with different prote-ases show extensive protection by DNA at the N-terminal duplicated con-served motif 1A⁄ 1B segment, and a well-folded dom

novel uracil-DNA-degrading factor

Ma´ria Puka´ncsik1, Ange´la Be´ke´si1, E´va Klement2, E´va Hunyadi-Gulya´s2, Katalin F Medzihradszky2,3, Jan Kosinski4,5, Janusz M Bujnicki4,6, Carlos Alfonso7, Germa´n Rivas7and Bea´ta G Ve´rtessy1

1 Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary

2 Proteomics Research Group, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary

3 Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA

4 Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Warsaw, Poland

5 PhD School, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland

6 Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland

7 Chemical and Physical Biology, Centro de Investigaciones Biolo´gicas, Madrid, Spain

Introduction

The nucleobase uracil is not a normal constituent of

DNA, although it provides the same Watson–Crick

interaction pattern for adenine as does thymine (i.e

5-methyl-uracil), and is actually used as the adenine-counterpart base in RNA Despite its usual absence, there are two physiological ways for uracil to appear

Keywords

cell death; DNA; nuclease; protein structural

modeling; uracil

Correspondence

B G Ve´rtessy, Institute of Enzymology,

Biological Research Center, Hungarian

Academy of Sciences, H-1113, Budapest,

Karolina u´t 29, Hungary

Fax: +36 1 466 5465

Tel: +36 1 279 3116

E-mail: vertessy@enzim.hu

(Received 2 April 2009, revised 16

December 2009, accepted 18 December

2009)

doi:10.1111/j.1742-4658.2009.07556.x

Uracil in DNA is usually considered to be an error, but it may be used for signaling in Drosophila development via recognition by a novel uracil-DNA-degrading factor (UDE) [(Bekesi A et al (2007) Biochem Biophys Res Commun 355, 643–648] The UDE protein has no detectable similarity

to any other uracil-DNA-binding factors, and has no structurally or func-tionally described homologs Here, a combination of theoretical and experi-mental analyses reveals the domain organization and DNA-binding pattern

of UDE Sequence alignments and limited proteolysis with different prote-ases show extensive protection by DNA at the N-terminal duplicated con-served motif 1A⁄ 1B segment, and a well-folded domain within the C-terminal half encompassing conserved motifs 2–4 Theoretical structure prediction suggests that motifs 1A and 1B fold as similar a-helical bundles, and reveals two conserved positively charged surface patches that may bind DNA CD spectroscopy also supports the presence of a-helices in UDE Full functionality of a physiologically occurring truncated isoform in Tribolium castaneum lacking one copy of the N-terminal conserved motif 1

is revealed by activity assays of a representative truncated construct of Drosophila melanogasterUDE Gel filtration and analytical ultracentrifuga-tion results, together with analysis of predicted structural models, suggest a possible dimerization mechanism for preserving functionality of the truncated isoform

Structured digital abstract

l MINT-7385914 : UDE (uniprotkb: Q961C4 ) and UDE (uniprotkb: Q961C4 ) bind ( MI:0407 ) by cosedimentation in solution ( MI:0028 )

Abbreviations

DmUDE, Drosophila melanogaster uracil-DNA-degrading factor; DmrcUDE, recombinant Drosophila melanogaster uracil-DNA-degrading factor; MQAP, model quality assessment program; TcUDE, Tribolium castaneum truncated uracil-DNA-degrading factor isoform; UDE,

uracil-DNA-degrading factor; UDG, uracil-DNA glycosylase.

in DNA: cytosine deamination and thymine

replace-ment Cytosine-to-uracil transitions via hydrolytic

deamination are among the most frequently occurring

spontaneous mutations These generate premutagenic

U:G mispairs [1,2] Thymine replacement by uracil can

occur if the cellular dUTP⁄ dTTP ratio increases, as

most DNA polymerases will incorporate either uracil

or thymine against adenine, based solely on the

avail-ability of the corresponding building block nucleotides

[3,4] Thymine-replacing uracil moieties are not

muta-genic, as they provide the same genomic information,

but may perturb the binding of factors that require the

5-methyl group on the thymine ring for recognition

There are also two mechanisms to ensure uracil-free

DNA: prevention and excision dUTPases prevent

uracil incorporation into DNA by removing dUTP

from the DNA polymerase pathway [5] Uracil in

DNA, produced by either cytosine deamination or

uracil misincorporation, is excised by uracil-DNA

gly-cosylases (UDGs) in the base excision repair pathway

[6,7] Among the different UDGs, the protein product

of the ung gene (termed UNG) is by far the most

effi-cient in catalyzing uracil excision [8] UNG is

responsi-ble for most of the repair process, as its mutation in

Escherichia coli, mouse and human has been found to

induce a considerable increase in uracil content [9–11]

Null mutations in the dUTPase gene (dut) result in a

nonviable phenotype that can be rescued by a second

null mutation in the ung gene The dut)ung) genotype

presents mass uracil incorporation into DNA [9,12]

Interestingly, an analogous situation, with

simulta-neous lack of dUTPase and UNG activities, arises in

Drosophila larvae under physiological conditions On

the one hand, the ung gene coding for the major UDG

enzyme is not present in the Drosophila genome [13]

On the other hand, it has been shown that the

dUTPase level is under the limit of detection in larval

tissues, and that the enzyme is present exclusively in

the imaginal disks [14] Simultaneous lack of UNG

and dUTPase may lead to accumulation of

uracil-substituted DNA in fruitfly larval tissues A specific

protein termed uracil-DNA-degrading factor (UDE),

which recognizes and degrades uracil-DNA, was

identified in Drosophila late larvae and pupae,

strengthening the hypothesis that Drosophila

melanog-aster may use uracil-DNA as a signal to switch on

metamorphosis-related cell death [15–17]

UDE is the first member of a new protein family

whose members recognize uracil-DNA It has no

glycosylase activity, and its sequence does not show

any appreciable similarity to those of other nucleases

or uracil-DNA-recognizing proteins [15] (Fig 1)

Sig-nificantly similar protein sequences were found only in

translated genomes of other pupating insects, but no structural or functional data have been published on any of these putative proteins In all of these sequences

of homologous proteins, four distinct conserved sequence motifs could be identified (motifs 1–4), the first of which is substantially longer and is usually present in two copies (motifs 1A and 1B) Comparison

of these motifs with motifs in UDGs does not offer any clue regarding the structure and function of UDE, as no apparent similarity could be observed (Fig 1B–E) [18] Investigation of this protein may therefore offer new insights into the physiological role and catalytic mech-anism of nucleases

To this end, in the present study we probed the domain organization of UDE from D melanogaster, expressed as a recombinant protein (DmrcUDE), by limited proteolysis, and revealed that a specific trun-cated fragment lacking the N-terminus may fold into a stable conformation Interestingly, we also identified such a truncated physiologically occurring UDE iso-form from the pupating insect Tribolium castaneum (TcUDE) [19] The TcUDE isoform lacks one copy of the N-terminal duplicated first motif We generated the respective segment from DmrcUDE by chemical cleav-age with hydroxylamine, and found that this truncated segment retains catalytic specificity and activity The structural results therefore offer an explanation for the physiological existence of the truncated isoform

De novo modeling was performed using rosetta, and

a 3D structural model was constructed for the tan-demly duplicated N-terminal motifs 1A and 1B The model suggests that both motifs comprise similar three-helical bundles, with the same topology and rela-tive orientation of a-helices A high content of helical secondary structure in UDE was also independently confirmed by CD The predictions, together with the domain organization studies, offer a model of DNA binding to an extended surface on the protein along the conserved motifs

Results

Identification of a physiologically occurring truncated isoform of UDE

blast searches indicated that UDE has detectable homologs only in pupating insects (Fig 1A) The mul-tiple sequence alignment shows four conserved motifs (Fig 1A,E) The first extended UDE motif is present

in two highly similar copies The UDE homolog from

T castaneum contains only one copy of motif 1, sug-gesting that lack of the first motif may still result in a functional protein (Fig 1A)

AUDG

UDGX

SMUG UNG

DRUDG

1 2 3

Common α/β fold

1A 1B 2 3 4

UDG families Motif 1 Motif 2 Motif 3

MUG/TDG hhhxGINPGL F/Y hhhFxG haVhPppSh

DRUDG xLxLLExPGP f VVhxLG xhxxxHPSh

UDE motifs 1A/1B GFKDxxxAxxTLxxLxxRDxpYpxxxhxGLhxxAKRVLxxTKxExKhxxIKxAhxxhEpaL

4 KxFpxcxxxPTxxHLxxIxWAYSxpxxKhK

UDE

UDG families B

A

D

E

C

Fig 1 Sequence alignment of UDE homologs in D melanogaster and T castaneum, and conserved motifs in UDE and members of the UDG superfamily (A) Alignment of D melanogaster and T castaneum UDE homologs Gray background: conserved motifs Red letters: strictly conserved residues (B) Evolutionary relationship and organization of conserved motifs among UDG proteins [18] Gray background: uracil-DNA-recognizing proteins present in D melanogaster (C) Organization of conserved motifs in UDE (D, E) Consensus sequences of UDG (D) and UDE (E) motifs Upper-case letters: conserved residues Lower-case letters: residues with conserved characteristics (h, hydro-phobic; a, aromatic; p, polar ⁄ charged) Nonconserved positions are indicated by x A conserved F ⁄ Y residue, overlapping with the uracil ring,

is invariably present C-terminal to motif 1 in UDGs Underlined Asp ⁄ Glu residues in UDG motif 1 are involved in catalysis; the underlined His

in UDG motif 3 is suggested to stabilize reaction intermediates Note the lack of detectable similarities between UDE and UDG motifs.

To confirm the in silico prediction of the UDE-like

protein product in Tribolium, extracts of the insect

larvae were investigated by western blot, using the

polyclonal antiserum produced against DmrcUDE As

expected from the high sequence similarity, the

antise-rum recognized the Tribolium protein (TcUDE) as well

(Fig 2) The blot clearly indicates that larval extract

from T castaneum contains a single protein that reacts

with the UDE-specific antibody This positive band is

found at a position corresponding to a much lower

molecular mass than that of DmrcUDE and that of the

physiological form of D melanogaster

uracil-DNA-degrading factor (DmUDE) The altered position of

TcUDE was in agreement with the genomic data

(Fig 1A), and led to the conclusion that the

physiolog-ically occurring TcUDE lacks the N-terminal segment

These results suggest that an isoform of UDE lacking

motif 1A may fold on its own, and may form a

func-tional protein

Domain organization studies using limited

proteolysis

To delineate the domain organization of the UDE

pro-tein more precisely, limited proteolysis experiments were

performed Three proteases with different specificities

were used Experiments were conducted with DmrcUDE

alone, and also in the presence of added DNA to study

potential DNA-binding protein segments

Trypsin was selected first, as the UDE protein con-tains many potential tryptic cleavage sites (i.e Lys and Arg residues) scattered throughout the sequence Fig-ure 3A indicates fast initial fragmentation leading to loss of 5–7 kDa fragments from either the N-terminus

or the C-terminus, or both This initial fragmentation

is not affected by the presence of DNA Flexibility of the N-terminal segment (residues 1–47) is also sug-gested by the drastic overrepresentation of basic resi-dues, leading to an extremely high pI (11.5) for this segment At later stages of proteolysis, DNA protec-tion is evident, as a specific fragment persists stably in the presence of DNA, whereas this fragment is rapidly degraded in the absence of DNA Several smaller frag-ments are produced in relatively large amounts in the absence of DNA, whereas these peptides are practi-cally absent in the presence of DNA The data suggest the presence of an inner folded core, which is sug-gested to participate in DNA binding, on the basis of DNA-binding-induced stabilization The large number

of potential tryptic cleavage sites prevented straight-forward identification of the fragments, observed

on SDS⁄ PAGE, by MS

For further characterization and localization of pro-tein segments involved in DNA binding to UDE, two additional sets of experiments were conducted, using highly specific chymotrypsin [20] and Asp-N endopro-teinase These enzymes have considerably fewer poten-tial cleavage sites in the protein In both cases, protection by DNA is again evident (Fig 3B,C) Figure 3B shows that, in the absence of DNA, initial chymotryptic cleavage removes a segment of about 9.6 kDa from UDE, whereas in the presence of DNA, the removed peptide is much smaller, around 3 kDa

MS analysis of the initially cleaved fragments revealed that the C-terminus remained intact, and the two pep-tide bonds most sensitive to chymotrypsin could there-fore be localized at the N-terminus at Trp10 and Tyr69 in the presence and in the absence of DNA, respectively (Fig 3D) DNA binding is therefore asso-ciated with significant protection at the Tyr69-Arg70 peptide bond located within the conserved motif 1A

In addition, DNA-binding-induced conformational changes are also reflected at the Phe104-Glu105 and Tyr311-Ile312 peptide bonds, which become exposed in the presence of DNA (Fig 3D)

To characterize the potential involvement of the C-terminal region of UDE in DNA binding, Asp-N endoproteinase was also used for limited proteolysis,

as the C-terminus of the protein is rather rich in Asp residues (Fig 3C,D) When it is digested by Asp-N endoproteinase, the primary cleavage removes a short fragment of about 3.4 kDa, independently of the

55 kDa

36 kDa

DmUDE Dm rc UDE TcUDE

1A 1B 2 3 4

1 2 3 4

DmUDE

TcUDE

Fig 2 Immunodetection of UDE homolog from T castaneum.

Western blot indicates that polyclonal anti-DmUDE serum

recog-nizes the UDE homolog from T castaneum that appeared at a

lower position than physiological DmUDE or DmrcUDE Lane 1:

D melanogaster larval extract Lane 2: purified Dm rc UDE Lane 3:

T castaneum larval extract.

presence of DNA This loss is in good agreement with

a C-terminal cleavage (at Asp333) leading to the loss

of 2.6 kDa; cleavage at the first N-terminal Asp

(Asp44) would remove a peptide of 6.6 kDa, which is

much larger than estimated from the gel

electropho-retic analysis It is evident that, in the absence of

DNA, additional cleavages can also occur, yielding

23–25 and 17 kDa polypeptides, as observed on

SDS⁄ PAGE Binding of DNA induces significant

pro-tection against all of these cleavages, except at the

Asp333 site, which shows the same highly exposed

character for Asp-N endoproteinase digestion in the

presence and in the absence of DNA

The results of proteolytic experiments are

summa-rized in Fig 3D It is obvious that the segment

encom-passing motifs 2–4 is a well-folded part of the protein,

even in the absence of DNA that lacks exposed

prote-olytic sites [despite the presence of numerous potential tryptic, chymotryptic and Asp-N sites (Figs 1A and 3D)] Motifs 1A and 1B, on the other hand, are signifi-cantly more prone to proteolysis, especially in the absence of DNA DNA binding provides significant protection against proteolytic cleavage along motifs 1A and 1B, indicating either DNA-binding-induced conformational changes or covering of otherwise exposed proteolytic sites by DNA binding to these segments

Motif 1A is dispensable for UDE function

To produce a specific truncated DmUDE isoform mimicking the physiologically occurring protein in

T castaneum, we selected a chemical agent, hydroxyl-amine, that cleaves peptide bonds exclusively between

36 kDa

28 kDa

17 kDa

11 kDa

55 kDa

w/o U-DNA w/U-DNA

MM 0´ 30´ 60´ 120´ 180´ 0´ 30´ 60´ 120´

His – tag

N111

36 kDa

28 kDa

17 kDa

11 kDa

55 kDa

w/o U-DNA w/U-DNA 0´ 60´ 180´ 300´ MM 0´ 60´ 180´ 300´

36 kDa

w/o U-DNA w/U-DNA

0´ 15´ 30´ 60´ 0´ 15´ 30´ 60´ MM

45 kDa

29 kDa

24 kDa

20 kDa

14.2 kDa

F104

Trypsin digestion

Chymotrypsin digestion

Asp-N proteinase digestion

B

D

Fig 3 Initial domain analysis of DmUDE by limited proteolysis (A) Tryptic digestion pattern Arrows indicate fragments that are preferen-tially produced in the absence of DNA; the star shows the detected position of stable fragment persisting in the presence of DNA (B, C) Limited digestion patterns obtained using high-specificity chymotrypsin and Asp-N endoproteinase The timescale of limited digestion and the presence or absence of added ligand are indicated at the top of the gel MM, molecular markers (D) Summary of cleavage sites identi-fied by MS Top row: chymotryptic sites Bottom row: Asp-N sites Solid arrows indicate cleavage sites that are similarly observable in both the presence and the absence of DNA Dashed arrows indicate sites protected in the presence of DNA Dotted arrows indicate cleavage sites detected only in the presence of DNA The cleavage site of hydroxylamine is marked with a bold arrow.

Asn and Gly [21] There is only one such peptide bond

in DmUDE, at Asn111-Gly112 (Fig 3D), located

between motifs 1A and 1B Figure 4A shows that, in

agreement with the previously determined exposed

character of the linker segment between motifs 1A and

1B, hydroxylamine cleaved the protein into an

N-ter-minal Met1–Asn111 and a C-terN-ter-minal Gly112–Glu355

fragment, as verified by MS The molecular masses of

the cleavage products are 14 and 28 kDa as calculated

from the sequence, whereas values of 16 and 32 kDa

were estimated from the SDS⁄ PAGE gels The

C-ter-minal fragment closely corresponds to the

physiologi-cal TcUDE isoform The presence of the N-terminal

His-tag on DmrcUDE allowed straightforward

separa-tion of N-terminal and C-terminal hydroxylamine-cleaved segments by Ni2+–nitrilotriacetic acid chroma-tography (Fig 4A)

To check whether the removal of motif 1A alters the specific function of the protein, we performed catalytic assays and electrophoretic mobility shift assays with the purified Gly112–Glu355 C-terminal fragment Fig-ure 4B shows that the C-terminal segment preserves catalytic activity and specificity for uracil-substituted DNA that do not depend on the presence or absence

of available divalent metal ions The gel shift indicates the DNA-binding capability of the C-terminal frag-ment, and also demonstrates the specific DNA-cleaving activity (Fig 4C)

N111

His-tag 1A 1B 2 3 4 Dm rc UDE

N-terminal M1-N111 C-terminal G112-E355

Intact

HA digested

Purified C-term

Intact G112-E355 M1-N111

0 ′ 30′ 60′ 120′ 31-mer 0 ′ 30′ 60′ 120′ 0 ′ 30′ 60′ 120′ 0 ′ 30′ 60′ 120′

G112-E355 Dm rc UDE

31-mer

0 ′ 30′ 60′ 90′ 0′ 30′ 60′

G112-E355 Dm rc UDE

G112-E355 Dm rc UDE Full-length Dm rc UDE

A

Fig 4 (A) Production and characterization

of the truncated UDE isoform Cleavage with hydroxylamine (HA) generates the expected fragments In the schematic repre-sentation, the single cleavage site at Asn111 between the 1A and 1B motifs is marked with an arrow Gel images show gelectrophoretic analysis of hydroxylamine cleavage and purification of the C-terminal motif to homogeneity (B) Electrophoretic mobility shift assay The concentration of

Dm rc UDE Gly112–Glu355 segment used in the experiment is given at the top of the lanes (lgÆmL)1) Uracil-DNA plasmid,

20 lgÆmL)1, was used in all mixtures (C–E) Truncated UDE lacking motif 1A retains uracil-DNA-degrading activity (C) Uracil-DNA

or control DNA linearized plasmid was incubated for the indicated time periods with truncated DmrcUDE (Gly112–Glu355 segment) Note degradation (as well as shift) of the uracil-containing DNA plasmid substrate (D, E) Activities of full-length UDE and Gly112–Glu355 truncated DmrcUDE constructs were compared using uracil-containing fluorescently labeled synthetic double-stranded (ds) and single-stranded (ss) oligonucleotide substrates (incubation times are indicated) Note the specific degradation product very close to the 31mer standard position, indicating that cleavage of the oligonucleotide only occurred at the uracil-containing position The catalytic activity of the truncated enzyme is still present, but

is detectable only on single-stranded substrate.

To clearly identify the cleavage site of the UDE

pro-tein and its truncated form on uracil-containing DNA

substrate, we performed cleavage experiments using

synthetic 60mer single-stranded and double-stranded

oligonucleotides, containing one single uracil moiety in

one of the strands, at the 32nd position The

uracil-containing strand was labeled with a fluorescent dye to

aid visualization of the reaction (Fig 4D,E)

Quaternary protein structure of full-length and

truncated proteins

To determine whether the absence of the N-terminus

has any effect on the quaternary structure organization

of UDE, the native molecular masses for the

full-length protein and the C-terminal fragment were

determined by analytical gel filtration The full-length

protein eluted at a position corresponding to 52 kDa,

which is somewhat larger than the full-length

mono-mer calculated molecular mass of 41.446 kDa This

alteration may indicate partial rapid equilibrium

dimerization and⁄ or the anomalous gel permeation

behavior may suggest that the proteins contain

signifi-cant amounts of natively unfolded, highly flexible

seg-ments To check this suggestion, we performed an

in silico analysis using several servers for

sequence-based prediction of structural disorder [22–24] The

results are shown in Fig 5, and indicate that the

dif-ferent predictors suggest, in agreement, considerably

high flexibility at the N-terminus and C-terminus, as

well as in the region between motifs 1A and 1B

Inter-estingly, the C-terminal Gly112–Glu355 fragment

eluted from the gel filtration column at practically the same position as observed for the full-length UDE, corresponding to 52 kDa As the calculated molecular mass of the monomeric Gly112–Glu355 fragment is

28 kDa, the elution profile strongly suggests that this fragment forms a dimer

Analytical ultracentrifugation was also applied to corroborate the results from the gel filtration studies The sedimentation equilibrium technique is reported to

be optimal for determining native molecular masses [25] In fact, our results with full-length DmrcUDE indicate that the determined molecular mass was 42.8 ± 2 kDa, in very close agreement with the mass calculated from the amino acid sequence (Fig 6) For the truncated Gly112–Glu355 construct, the deter-mined native molecular mass was 49 ± 1.2 kDa, cor-responding rather closely to a dimer of the truncated segment (for which the calculated masses are 28 kDa for the monomer and 56 kDa for the dimer) These results, in agreement with the gel filtration data, argue for a native monomer of the full-length protein and a native dimer for the truncated construct

Sedimentation velocity experiments revealed that full-length DmrcUDE has a main sedimenting species (82% of the loading concentration) with a standard sedimentation value of 2.6S ± 0.1S, which, together

–0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

RONN IUPred DISOPRED

Number of amino acids

Fig 5 Disorder profile of Dm rc UDE The plot shows sequence

position against probability of disorder Segments of the sequence

at the N-terminus and C-terminus and between motif 1A and motif

1B were classified as disordered by three predictor programs

( IUPRED , RONN , and DISOPRED ).

–0.03 0.00 0.03

0.0 0.2 0.4 0.6 0.8 1.0

Sedimentation coefficient (S)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

A280

Radius (cm) Fig 6 Determination of UDE oligomer status by analytical ultracen-trifugation Top panel: sedimentation equilibrium gradients of 0.53 mgÆmL)1 full-length DmrcUDE (s) and 0.8 mgÆmL)1 for

Dm rc UDE Gly112–Glu355 (h) at 13 400 g as described in Experi-mental procedures The solid line shows the fit of the experiExperi-mental data to single ideal species Bottom panel: residual distribution as a function of the sedimentation distance (this plot corresponds to the difference between the experimental data and the fitted data for each point) Inset panel: sedimentation coefficient distributions of full-length Dm rc UDE (solid line) and Dm rc UDE Gly112–Glu355 (dashed line).

with the sedimentation equilibrium data, is compatible

with a protein monomer whose hydrodynamic

behav-ior deviates from the expected for a globular species

[calculated frictional ratio (f⁄ f0) = 1.6]; the rest of the

protein sediments as faster oligomeric species The

truncated Gly112–Glu355 protein construct showed

significant polydispersity, with main peaks at 2.5S,

4.3S, and 6.0S, representing approximately 70%, 20%,

and 6%, respectively, of the loading concentration

The 2.5S peak is compatible with a protein globular

monomer (f⁄ f0= 1.3) These data argue for potential

monomer self-association into dimers and higher-order

oligomers

Structure prediction of UDE reveals a

pseudosymmetrical arrangement of two a-helical

bundles

For structural prediction, the DmUDE full-length

sequence was submitted to the genesilico

metaserv-er[26], which is the gateway providing a unified

inter-face to several servers for secondary and tertiary

structure predictions The analysis of predictions of

domain composition suggested that UDE contains an

N-terminal helical region of approximately 30 residues

and at least three structural domains corresponding to

motifs 1A and 1B, and the C-terminus, encompassing

motifs 2, 3 and 4 The C-terminus of 40–50 residues

and the loop connecting motifs 1A and 1B (between

residues 109 and 137) are predicted to be mostly

dis-ordered All three domains are predicted to be mainly

helical, although the secondary structure predictions

for the third domain were uncertain, as there was no

agreement between alternative servers

The fold recognition analysis did not reveal any

con-fident matches with known protein structures,

suggest-ing that the UDE 3D structure may exhibit a novel

fold Therefore, to predict at least partially the tertiary

structure of UDE, we performed de novo modeling of

the region encompassing motifs 1A and 1B, using the

rosetta program [27] In total, about 500 000

differ-ent models (also known as decoys) were generated,

and 10% of the lowest-energy structures were clustered

on the basis of their similarity The representatives of

the best clusters were refined with the rosetta full

atom refinement protocol, and scored with the model

quality assessment programs (MQAPs) proq [28] and

metamqap [29] Evaluation of the largest clusters

revealed that both motifs 1A and 1B comprise similar

three-helical bundles, with the same topology and

rela-tive orientation of the helices Nevertheless, the top

clusters differed in relative orientation of the two

heli-cal bundles to each other (data not shown) Among

these clusters, one single cluster contained the specific topology that exhibited pseudosymmetrical orientation

of the two motifs Importantly, members of this cluster exhibited low energy levels and were well scored by MQAPs (proq – predicted LGscore in the range 1.2–3.2, and metamqap – predicted rmsd in the range 3–4.2 A˚, for the five lowest-energy representatives of the cluster), which indicates a high probability that they resemble the currently unknown native structure Figure 7 depicts the predicted model in several different orientations The two homologous motifs (1A and 1B) form a four-helix bundle interaction surface (Fig 7A,B) On the surface of the model, a well-con-served, positively charged surface is well defined This may serve as the nucleic acid-binding surface, in agree-ment with the limited proteolysis data

Estimation of secondary structural elements by

CD spectroscopy

To verify structural predictions, CD spectroscopy mea-surements were performed, as CD spectra in the

far-UV wavelength (190–240 nm) range are very indicative

of different secondary structural elements [30] Spectra

of the intact protein and of the C-terminal fragment Gly112–Glu355 showed double negative maxima at

208 and 222 nm, which are characteristic for the pres-ence of a-helices (Fig 8) Quantitative evaluation of the spectral data was performed with k2d and selcon [24,31,32] The estimated percentages of protein sec-ondary structures from CD spectra reveal 37% a-heli-ces and 18–26% b-structure

Discussion

The potential signaling role of deoxyuridine moieties in genomes of pupating insects was first suggested by Deutsch et al [16], on the basis of the lack of UDG activity in these insects The hypothesis stating that uracil-DNA might be present transiently in larval stages and that its degradation at the end of larval stages may contribute to cell death during metamor-phosis was much debated, owing to independent find-ings from several laboratories showing the presence of UDG activity in some developmental stages of Dro-sophila [33–37] This debate was resolved by the fully annotated Drosophila genome, which clearly indicated the lack of the major UDG gene ung but the presence

of several other genes that encode catalytically much less efficient UDGs The absence of dUTPase in larval stages [14] and our recent discovery of the strictly regu-lated UDE [15] reinforced the hypothesis on the possi-ble role of uracil-DNA in Drosophila and suggested a

role for UDE in programmed cell death during meta-morphosis Functional analysis of UDE identified this protein as a novel uracil-recognizing factor [15], with

no similarities to either UDGs [18] or the Exo-III⁄ Mth212 nuclease [38] Multiple sequence alignments

of UDE homologs from all available pupating insect genomes indicated the presence of conserved motifs in most species, with the same distribution (Fig 1) The UDE homolog in T castaneum lacks one copy

of the N-terminal duplicated first motif (Figs 1 and 2) TcUDE showed reactivity with the antiserum produced against DmrcUDE, suggesting that the truncated TcUDE isoform is a well-folded UDE-like protein It was also observable on the blot that the physiological forms of the proteins from both Drosophila and Tribo-lium extracts were detected at much higher electro-phoretic positions than expected from the calculated molecular mass values: molecular masses estimated

–10 000

–8000

–6000

–4000

–2000

0

2000

4000

6000

Intact Dm rc UDE

G112-E355 Dm rc UDE

Wavelength (nm)

ΘMRE

2 ·dmol

Fig 8 CD spectra of intact UDE (solid line) and C-terminal

frag-ment (dashed line) confirm the presence of a-helices MRE, mean

residue molar ellipticity.

Orange – strictly conserved

Green – variable

Cartoon model colored

by motifs

(blue and red – protease

cleavage sites)

Cartoon model colored by sequence conservation

Surface model colored by sequence conservation

Surface model colored by electrostatic potential

Motif 1A Motif 1B

–3 kT/e + 3 kT/e

Orange – strictly conserved

Green – variable

Fig 7 Structural model of DmUDE duplication fragment Structures are shown in two views: front (upper panel) and top (bottom panel) (A) Cartoon representation Duplicated motifs 1A and 1B are colored green and orange, respectively, and the nonconserved linker is colored gray Peptide bonds protected from proteolytic cleavage on DNA binding are colored blue The peptide bond between residues 104 and 105, cleaved only on DNA binding, is colored red Note that the duplicated fragments are only approximately symmetrical, as the model is of low resolution and the local conformation of the backbone is uncertain (B, C) Sequence conservation mapped onto the ribbon diagram (B) or the molecular surface (C) (conserved residues are colored orange and yellow; variable residues are colored green) (D) Electrostatic potential mapped onto the molecular surface (positively and negatively charged regions are colored blue and red, respectively) Arrows indicate the positively charged conserved patches that may accommodate DNA.

UDE sequence

BLAST

Multiple sequence alignment

T cas predicted

protein product

Western blotting

Secondary structure prediction

Modeling

Identification

of conserved

surface patches

Mapping

of electrostatic potential

Prediction

of DNA binding site

Limited proteolysis

Peptide identification

by MS Trypsin

Asp-N endoproteinase Chymotrypsin

Hydroxylamine cleavage

Analysis

of C-ter fragment

DNA binding

by EMSA

DNA cleavage assay

Quaternary structure

by gel flitration

Theoretical analysis Experimental analysis

Domain organization

Analytical ultracentrifugation

Circular dichroism

Fig 10 Flowchart scheme of bioinformatics and experimental approaches.

Motifs 2,3,4 Motifs 2,3,4 Motifs 2,3,4

Motif 1 B

Motif 1 A Motif 1 Motif 1

C

Fig 9 Structural models of DmUDE pseu-dodimer (A) and TcUDE dimer (B) Struc-tures are shown in cartoon representation and colored by motif (motif 1A in DmUDE and motif 1 in TcUDE, dark red; motif 1B, dark gray; nonconserved segments, light gray) Residues 1–11 of TcUDE are not shown (the conformation of this fragment is very uncertain) C-terminal parts correspond-ing to motifs 2, 3 and 4 are shown schemat-ically only (C) Alignment between motif 1 residues for DmUDE and TcUDE Identical and conserved residues are colored red and green, respectively The helical prediction is indicated Note the numerous conserved hydrophobic and polar residues that may form the dimerization surface.