Functional changes in temperature-sensitive mutants of the adenovirus single-stranded DNA-binding protein are accompanied by structural alterations.

0022-538X/92/010480-09$02.00/0

Copyright X) 1992, American Society forMicrobiology

Functional

Changes

in

Temperature-Sensitive

Mutants

of

the

Adenovirus

Single-Stranded

DNA-Binding Protein

Are Accompanied

by Structural Alterations

MASAYOSHITSUJI ANDGEOFFREY R. KITCHINGMAN*

DepartmentofVirology andMolecularBiology, St. JudeChildren's ResearchHospital,

Memphis,

Tennessee

38101-0318

Received5 August1991/Accepted9 October1991

Adenovirus requires the virus-encodedsingle-strandedDNA-binding protein(DBP)toreplicateits DNA.We

have previously shown (M. Tsuji, P. C. van derVliet, and G. R. Kitchingman, J. Biol. Chem.

266:16178-16187, 1991) that theinabilityof threetemperature-sensitive(ts)mutantDBPs

(Ad2+ NDlts23, Ad2ts111A,

and

Ad5ts125)tosupport DNA replication at thenonpermissive temperaturewasassociated withimpaired ability

to bind to DNA. In thisstudy, we examined thesemutantproteinsfor structural alterations thatmightbelinked to the functional changes. Allthree ts mutants, butnot the wild-type protein, showed different proteolytic cleavage patterns before and after heating at40°C (the nonpermissive temperature), suggesting a possible conformational change during heating. The Ad2+NDlts23 and Ad2ts111A DBPs have single amino acid changes located in a putative zincfingersubdomain(positions282 and280).Inthepresence of zincions,these ts mutants showed significantly increased resistance to inactivation at 40°C.

Surprisingly,

however, the

stabilizing effectof zinc was also observed with theAd5ts125 DBP,whichcontainsamutation locatedmorethan

100amino acids from the zincfinger.Other related metalions,such ascobalt,cadmium,andmercury,did not protect the ts DBPs from inactivation at40°C. These resultsindicatethatfunctionalchangesof the ts DBPs in DNAreplication and DNA binding are accompanied by structural alterations in theproteinand thatzinc and

themetal-binding subdomainmayplay animportantrolein thestructureand/or functionofthe DBP.

One of theviralproteins expressed

during

theearly phase

of adenovirus infection isa single-stranded (ss)

DNA-bind-ing protein(DBP). Thisproteinis produced in large

quanti-tiesandis localized predominantly in the nuclei of infected

cells. The DBP performs multiple functions during the

infectious cycle, includingsupportof viralDNAreplication

(reviewed in reference 10), regulation ofviralgene

expres-sion at both the transcriptional (24) and the

posttranscrip-tional (3) levels, determination of host range (14), and

involvement in the virus assemblyprocess(25). Theprotein

binds preferentially to ssDNA with no apparent sequence

specificity and binds to both double-stranded DNA (8, 29,

32) andRNA(1,5, 30). The apparent molecularweightof the

DBP, as estimated by sodium dodecyl sulfate

(SDS)-poly-acrylamide gel electrophoresis (PAGE), is 72,000 (13), but

theprotein is actually composed of529 aminoacids,which

gives a calculated molecular weight of 59,049 (16). This

discrepancy

isprobably dueto ahighdegree of

phosphory-lation (13) andtotheproline-richnature of the protein (16),

leadingtothe anomalous migrationin SDS-containinggels.

Biochemical studies have revealed the presence of two

structurally andfunctionally distinct domainsinthe protein.

Mild chymotrypsin digestion splits the protein into highly

phosphorylated 26-kDa amino-terminal and nonphosphory-lated 44-kDa carboxyl-terminal fragments (13). The

car-boxyl-terminal domain is capable of binding to ssDNA (13)

and RNA (5) and is fully functional in supporting DNA

replication in vitro (2, 9). Genetic studies have identified

amino acid residues important for nuclear localization (21)

andfor hostrange (16) in theamino-terminaldomain of the

protein. Three highly conserved regions (designated CR1,

CR2,

and CR3) within the carboxyl-terminal domain have

*Correspondingauthor.

been found by comparative sequence analysis (12) among theDBPs of various human adenovirus serotypes. Some of the mutations introduced in CR2 (amino acids 322 to 330)

andCR3(aminoacids464 to 475) substantiallydecrease the

protein's ability to bind to ssDNA and to support

adeno-associated virus DNA replication in vivo (22, 23) without

affectingits in vivostability, phosphorylation,orsubcellular

localization. Thus, these two regions of the DBP may be directly involved in DNAbinding.

Three temperature-sensitive (ts) adenovirus mutants

(Ad2+ND1ts23, Ad2tslllA, and Ad5tsl25) that are

defec-tive in DNA replication at the nonpermissive temperature have been isolated, identifying two other regions of the carboxy-terminal domain important for the functions of the protein. The Ad2+NDlts23 (17) and Ad2tslllA (27) muta-tions are in closeproximity (a Leu-to-Phe change at amino acid 282 and aGly-to-Val change at amino acid 280, respec-tively), and these amino acids are flanked by -His-X-Cys-and -Cys-X-His-, which has the potential to coordinate a zinc ion (37) in a fashion similar to that found in "zinc finger" proteins (4, 7). These mutant proteins appear to be stable and properly phosphorylated in vivo at the nonper-missive temperature (27, 31). TheAd5tsl25DBP (6), which has a Pro-to-Ser change at amino acid 413 (16), has been used to identify most of the DBP's functions in RNA metabolism, as well as its function in DNA replication. The mutantprotein produced in vivo at the nonpermissive tem-perature ispoorly phosphorylated(19)andrapidlydegraded

(11, 34), suggesting that the mutation is in a region that is

important for theprotein'sstability. Interestingly, however, functionaldefects caused by thismutation can be corrected

phenotypically by secondary mutations at amino acid 347,

352, or 508 (15,26).Collectively,theseDBP mutantsidentify

fourregions importantforproperfunctioningof the protein.

Wehavepreviously demonstratedthat the three ts mutant

480

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TEMPERATURE-SENSITIVE ADENOVIRUS DNA-BINDING PROTEINS 481

DBPs lose the ability to bind to oligonucleotides when

heated at the nonpermissive temperature, suggesting that

their inability to support DNA replication results from

impaired DNA-binding activity (33). A key question that

remained to be answered was whether the mutations are

located in the sites of the protein that directly interact with ssDNA, thereby causing a functional change without altering the overall protein structure, or whether heating at the

nonpermissive temperature induces a structural alteration

that affects DNA binding and perhaps interaction with the

adenovirusDNA polymerase (20). To address this question,

weexamined the proteolytic cleavage patterns of the three ts mutant proteins before and after heating at the nonpermis-sive temperature and found differences that were indicative ofapossible conformational change. We also investigated a

possible role of zinc ion and the putative metal-binding

subdomain (amino acids 273 to 286) inthefunctions of the

DBPbecause boththeAd2+NDlts23 andAd2ts1llA

muta-tions arelocated within thisregion. In thepresence of zinc,

these two mutant proteins, as well as the Ad5tsl25 DBP,

retainDNA-binding activity over significantly extended

pe-riods at the nonpermissive temperature. MATERIALS AND METHODS

DBPs. The wild-type and three ts mutant DBPs were

purified (29) from KB cells infected with AdSdl301,

Ad2+NDlts23, Ad2tslllA,andAdStsl25with the

modifica-tionsdescribed previously (33).

Proteolytic digestions. DBP preparations were diluted to

0.5mg/ml in TEMG buffer(10 mMTris-HCl[pH8.0], 1 mM

EDTA, 2 mM 2-mercaptoethanol [2-ME], 10% glycerol)

containing 100 mM NaCl. These samples were either

di-gesteddirectly orheated to 40°C for 30 min prior to

diges-tion. Reactionswere set up onice by mixing 2

R1

of DBPs (1

,ug) and4 ,ul ofproteolytic enzymes (1 ng/,lp) in thebuffers

described below. Tosylsulfonyl phenylalanyl

chlorometh-ylketone-treated trypsin and tosyl lysine chloromethyl

ke-tone-treated chymotrypsin (both from Sigma) digestions

wereperformed in bufferP (40 mMTris-HCI [pH 8.0], 1 mM

EDTA, 2 mM 2-ME, 10%glycerol)containing0.5 MNaClat

30°C for15and 20min,respectively,andstoppedwith 0.5p.1

of100 mM phenylmethylsulfonyl fluoride. Staphylococcus aureus V-8 protease (Pierce) digestions were done in 100

mM phosphate buffer (pH 7.8) at 30°C for 30 min and

terminated by

heating

at

90°C

for 5min.

Thermolysin

(Sig-ma)digestionsweredone in20 mMTris-HCl(pH8.0-2 mM

CaCl2-1.4

mM 2-ME-5%

glycerol

at

30°C

for 15 min and

stopped with 2 ,ul of50 mM EDTA.

Papain

(Sigma)

diges-tions were performed in buffer P at

30°C

for 15 min and

stopped with 1

pl

of50 mM iodoacetamide. Proteinase K

(BethesdaResearch Laboratories) digestionswerealso

per-formed inbufferPat

30°C

for15min and

stopped

with 0.5 ,ul

of100 mM

phenylmethylsulfonyl

fluoride.

Proteolytic

frag-ments were separated by SDS-PAGE

(18)

and visualized with the Bio-Rad Silver Stain kit.

Gelmobilityshiftassay.Detailsof the

gel

shiftassay

using

an 84-mer DNA have been described

previously (33).

Briefly, the wild-type and ts DBPs, either untreated or

treated as described

below,

were mixed with

specified

amounts of

32P-labeled

84-mer DNA.

Samples

were incu-bated on ice for at least 60 min and then

electrophoresed

througha1%agarose

gel

in TBE

(45

mM

Tris-borate,

1mM

EDTA [pH 8.3]). Gels were

partially

dehydrated

and

ex-posed to Kodak XAR film with Cronex

Lightning-Plus

intensifying screens.

Stabilizing effect of DNA. The DBPs (200 ng) in TEMG buffercontaining 20 mM NaCl and 200

jig

of bovine serum albumin per ml were either heated to 40°C for various periods and then mixed on ice with 5 ng of 32P-labeled 84-mer DNA or mixed first with 5 ng of 32P-labeled 84-mer DNA onice and then incubated at 40°C for various periods. Freeandprotein-bound DNAs were separated at 4°C by the

gelmobility shift assay.

Stabilizing effect of antibody. A monoclonal antibody against DBP (B6 antibody in reference 28) was purified from hybridoma culture supernatants by affinity chromatography

by using an anti-mouse immunoglobulin G (IgG)-Sepharose

4B column. Four sets of reactions were carried out with 200 ngof DBPs in TEMG buffer containing 20 mM NaCl and 200 ,ugof bovine serum albumin per ml. In the first, DBPs alone were incubated at 40°C forvarious periods and then mixed with5 ngof

32P-labeled

84-merDNA. In the second and third

sets of reactions, DBPs were mixed with 200 ng of either

unimmunized mouse IgG (Sigma) or purified B6 IgG, the

mixtureswereheated at40°C for various periods, and 5 ng of

32P-labeled 84-mer DNA was added. In the fourth assay,

DBPs were heated at 40°C for various periods and then

mixed with200 ngofB6IgG and S ngof32P-labeled 84-mer

DNA. Reactions were analyzed at 4°C by the gel mobility

shift assay.

Effects of various metal ions on DNA-binding activity of

DBP. Solutions (15 p.l) containing 250 ng of DBP, 10 mM

Tris-HCl(pH 8.0), 1 mM EDTA, 2 mM 2-ME(omittedwhen

CoS04 was used), 20 mM NaCl, 0.2 mg of bovine serum

albumin per ml, 10% glycerol, and either zinc acetate,

CoS04, CdCl2, HgCl2, MnCl2, or MgCl2 at 2 mM were

incubated at 40°C for the periods indicated in the figures.

Sampleswerechilledonice,and EDTA wasadded to a final

concentration of2.5 mM. DNA-binding activitywas tested

at 4°C with 5 ng of 32P-labeled 84-mer DNA in the gel

mobility shiftassay.

EM. Samples (10 ,ul)for electron microscopy (EM)

con-tained 0.1 ,ug ofM13mpl8ssDNA and 4

jig

ofDBPs(either unheated or heated at 40°C for30 min) in 10 mMTris-HCl

(pH 8.0-1 mM EDTA-2 mM 2-ME-20 mM NaCI. The

sampleswereeitherdiluted with90p.l of 50% formamidein

100 mMTris-HCI (pH8.0-10mMEDTA, mixed with 2.5,ul

of 0.5% cytochrome c, and spread on distilled water or

dilutedwith 90 p,l of0.25 Mammoniumacetate, mixed with

2.5

,u1

of 1% cytochrome c, and spread onto a 0.025 M

ammonium acetate hypophase. Materials on the surface

were picked up with carbon-coated grids, stained with

alcoholic uranyl acetate, rotary shadowed with

platinum,

and examinedbyaPhilips 301 EM. RESULTS

Proteolytic digestion patterns. We

previously

demon-strated (33) that ts DBPs

Ad2+NDlts23,

Ad2tslllA,

and

AdStsl25 have wild-type

activity

at

30°C

in both the

oligo-nucleotide-binding and the in vitroDNA

replication

assays

butirreversibly lose bothactivities

by

heating

at

40°C.

The

mechanism

underlying

this change could be either a small

structural change confined to the

DNA-binding

site in the

protein or a

global

conformational

change

that makes the

protein no longer functional. One method of

distinguishing

between thesetwo

possibilities

isexaminationof themutant DBPs before and after

heating

at

40°C

for

susceptibility

to

proteolyticenzymes, asthe

accessibility

ofanenzymetoa

potential cleavage

site would differas a

protein

undergoes

a

conformational

change.

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Chymotrypsin

Trypsin

V8

protease

M

wt

tslllA ts23 ts125

Hdeating

r- r-, r--

r-at

400C 30'

+

-97

-66

6-M

wt

tsIllA

ts23

ts125

_ +-_ + -+ -+

M

wt

tslllA

ts23 tsl25

_-+

_-+ _+-_

+

no_ W.. - N_ _ - we

,,--a

urn

_

m - -_

so

40 mm

Thermolysin

_ " -"

_m _wo&do

Papain

NW

to _40a

m

so

Proteinose

K

am a_ -_

so

FIG. 1. Proteolytic digestionpatternsofwild-type (wt)and threetsmutantDBPs. The DBPs(1 ,ug,either unheatedorheated at40°Cfor

30min)weredigestedwitha1/250(wt/wt)dilution ofenzymeat30°C,and theproducts wereanalyzed bySDS-PAGE. Lanes Mcontained

markerproteins, whose sizesaregivenonthe left in kilodaltons.

Thewild-type and threemutantDBPs, either unheated or

heatedat40°C for 30min, weredigested separately with six

proteolyticenzymeshavingarangeofsubstratespecificities,

fromhighlyspecific (trypsin and V-8 protease)tononspecific (proteinase K). With each of the six enzymes, all three ts

mutantproteins yieldedatleastonecharacteristic band that was different between the heated and unheated samples, while the wild-type protein produced identical digestion patterns under thetwoconditions (Fig. 1). For chymotryp-sin, V-8 protease, trypsin, and thermolysin, lower-molecu-lar-weight peptide fragments were produced when the un-heated but not heated DBP was used as a substrate,

suggesting thatacleavage sitewasrendered inaccessible by heating. Withpapain and proteinase K, digestion of the DBP to lower-molecular-weight fragments was observed follow-ing heatfollow-ing, indicatfollow-ing increased accessibility to these

en-zymes. Thus, the ts mutants, butnotthe wild-type protein, probably undergo a conformational change at the nonper-missive temperature. This does not necessarily mean that theproteinsaretotally denatured: thetsDBPs thathad been heatedto40°C yielded specificcleavage fragments (Fig. 1), whereas those heated to 90°C for10 min did nothave such specific fragments, presumably because ofa more random conformation (data not shown). The unheated ts mutant

DBPs and those heated at 30°C for 30 min had digestion patterns similartothatof wild-type DBP (datanot shown), suggesting that the mutations do not significantly alter the

proteins' overallstructuresaslongastheyareproducedand kept atthe permissivetemperature. Interestingly, digestion of the unheated ts DBPs with the less specific proteolytic

enzymes papain and proteinase K yielded a predominant cleavage fragmentwithamolecularweightofapproximately 39,000. The production of such a fragment suggests the

presence of a rigid, compactly folded region within the protein, probably corresponding to a part of the carboxyl-terminal domain. Loss of thisfragment with heatingto40°C indicates that the mutations are located at sites that are

crucial for maintenance of the compactly folded structure. Stabilizationofts DBPsby DNA and antibody. Ifa struc-tural change in the mutant proteins is the cause oftheir ts phenotype, interaction of the DBP with other macromol-eculesmight interferewithconformationalchanges, protect-ing the proteins from functional inactivation atthe

nonper-missive temperature. Two macromolecules, an 84-mer ssDNA and an anti-DBP monoclonal antibody (B6), were

chosento test thishypothesis.

The wild-type and three tsDBPs wereheatedat 40°C for various periods and then mixed with 32P-labeled 84-mer DNA orfirst mixed with 32P-labeled 84-merDNA and then incubated at40°C. The DBP-DNA complex and free DNA wereseparatedat4°C byagarosegelelectrophoresis (Fig. 2). The ts DBPs existing free in solution were all rapidly inactivated at 40°C, but the proteins that hadboundto the 84-mer DNAremained associated withthe DNAat40°C for 31

-21.5- II 14.4 -

-97

66 t

43

31

21.5- _m

14.4 -_

amam 6m- MO-am--o

43- IW

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TEMPERATURE-SENSITIVE ADENOVIRUS DNA-BINDING PROTEINS 483

A B C

Inc. time r- I

1-i

I l

at 400 C 0 5 10 20 10 20 40 60 80 (min)

_ _ .. _ __- _. Al

DBF DNA

- _

__

_ _Am

A B - C D E

-,- - IrIC 1

Inc. time

at400C 0 1020 30 40 01020 3040 0 1020 304010203040

(min) t

DNA-DBP-Ab- iI I

DNA-DBP-iJIItS -_

wt

freeDNA-

0

to IIA

DNA-DBP-Ab-- t " DNA-DBP-*

0

free

DNA-

*

tea

DNA-DBP-Ab-DNA-DBP- *

seW'.

50d4.

SO

*-

_{freeDNA- _ M S_}

IIIr 11 Tl

Inac4tOnC

0 5 10 1520 0 5 10 15 200 5 10 1520 5 10 1520

(mnts

f52s

DNA-DBP-Au

-DNA-DBP -

6

0

a

aM

_~~~4

fa

0

ts125

FIG. 2. Gelmobility shift assay showing stabilization of ts DBPs byDNA. The DBPs(200 ng)wereeither incubatedat40°C forthe

indicated periods and mixed with 5 ng of32P-labeled84-mer DNA (A) or mixed with 5 ng of 32P-labeled 84-mer DNA and then incubated at 40°C for the indicated periods (B). Mixtures were

subjected toagel mobility shift assay at4°C. Lanes C contained

32P-labeled84-merDNAonly. wt, wild type; Inc., incubation.

aslongas 80min, indicatingthatbindingto DNArendered

the ssDNA-binding activity of the ts DBPs resistant to

thermalinactivation.

To examinethe effect ofantibody, DBPsalone or mixed

with either unimmunized mouse IgG or purified anti-DBP

antibody were heated at

40°C

forvarious periods and their

DNA-binding activities were examined at 4°C by the gel

mobility shiftassayusing 32P-labeled 84-mer DNA(Fig. 3).

Binding of antibodytothe DBPresultedin afurther shift of

free-DNA-

-

-

-FIG. 3. Gel shift assay showing stabilization of ts DBPs by an anti-DBPantibody (Ab). Four sets of experiments were carried out. DBPs alone were incubated at 40°C for the indicated periods (A). DBPsmixed with unimmunized mouse IgG were heated at 40°C (B). DBPs bound to B6 anti-DBP antibody were heated at 40°C (C). DBPs were incubatedalone at 40°C for the indicated periods and then mixed with B6antibody (D). DNA-binding activity was mea-suredat4°C by the gel mobility shiftassaywith32P-labeled84-mer DNA. Lanes E contained 32P-labeled 84-mer DNA only. Inc., incubation; wt, wild type.

themobility ofthe DNAband. The B6anti-DBP monoclonal

antibody did not interfere with binding of DBP to DNA.

Thus, the epitope recognized by the antibody, which has

beenmapped between amino acids 170and270ofthe DBP

(23), does not appear to be the site that directly interacts

with ssDNA. The ts DBPsbound to the antibody retained

their DNA-binding

activity

at

40°C

for significantly longer

periods thandid the unbound DBPs. The

antibody

had no

effectwhen itwas addedtothe ts DBPsafterthey had been

incubatedat40°C for30 min.

EMof DBP-M13 DNA complexes. We

previously

showed that thets DBPs lost the

ability

to bindto

oligonucleotides

after heating at

40°C

but retained some

binding

activity to

larger ssDNA molecules, such as M13 DNA

(33).

This

observation led us to

speculate

that the ts DBPs, when

free DNA

ts

ilA

ts

23

. _

v*a

a

...

040000

0

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J. VIROL.

484 TSUJI AND KITCHINGMAN

v~~ ~ ~ ~7~ ~te2F 1' 4: ' ~ -< , 4.:. ;

4: F -.s

Fy,ff 4

A * S 4:4 . F' ,* R

;S¢j,

S.' o!.1' * CA

4F 't

're*

BS*' 44 v Z ts

<d

'SC s

e.;'<...7.g~';',S-;G,"''...

p; Fb / ;' - 2

Wsrik

°

'p0t' - ;'S

#Jt W -LW'SS.>'w_,~

*4.~~~~~~~~~~

S~~~~

ttF "ar. i 't '44 ; ' ' '

44 "A~~~~~~~~~~~~~~~~~~~~~~~~~~~~A

St.,.

FIG. 4. Ad5tsl25 DBP-M13 DNAcomplexes observed by EM.(A) M13 DNAonly. (B) A4-,pgsample ofunheatedAd5tsl25DBP plus 0.1 ,ugof M13DNA. (C) A4-,ug sample ofAd5tsl25 DBPheated at40°Cfor30minand then mixed with0.1 pugofM13DNA.

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TEMPERATURE-SENSITIVE ADENOVIRUS DNA-BINDING PROTEINS 485

wt tsIllA ts23 ts125

Inc. time Zn(-) Zn(+) Zn (-) Zn (+) Zn (-) Zn(+) Zn

(-H

Zn (+)

at40CC 0 5 10 2030 10 20 40 60 0 5 lO 20

30'0

10 20 30 40 0 5 10 20 30 0 lO 20 30 40 0 5 1020 30 0 10 20 30 40

(min)

DBP-DNA-

**'ows

s.

free DNA- _rn

FIG. 5. Stabilizing effect of zinc on the DNA-binding activity of ts DBPs. DBPs (250 ng) were heated at40°Cfor various periods in 10 mM Tris-HCl(pH

8.0)-i

mM EDTA-2 mM 2-ME-20 mM NaCl-200 ,ug of bovine serum albumin per ml-10% glycerol or in the same buffer containing 2 mM zinc acetate, mixed with 5 ng of32P-labeled 84-mer DNA, and electrophoresed at 4°C through a 1% agarose gel. Inc., incubation; wt, wild type.

heatedto the nonpermissive temperature, bind to ssDNA in

anaberrant fashion.

To detect any alterations in the DNA-binding mode,

complexeswereformed betweenthe DBPs (either unheated

orheatedat40°C for30min) and M13 ssDNA at a DBP/DNA

(wt/wt) ratio of 40 and examined by EM. The complexes

were spread either from 50% formamide onto water or from 0.25 Mammoniumacetate onto 0.025 M ammonium acetate.

Typicalobservations are presented in Fig. 4. The M13 DNA

moleculeswere readily visualized as open circles (Fig. 4A),

whereas theAd5tsl25DBP-M13 DNA complexes were very

faint andhadslightlydistorted structures (Fig.4B).The poor

contrastmay bethe resultof interference by the DBP with the cytochrome c coating of DNA. Structures similar to

those in Fig.4B were alsoobserved withthe other unheated

ts DBPs and the wild-type protein. Interesting

structures-large, marblelike aggregates-were seen in the complexes

made with theAd5tsl25DBPheated to 40°C and M13 DNA

(Fig.4C). SimilarDNA-proteinaggregates wereformedwith

the heatedAd2+NDlts23 andAd2tslllADBPs but not with

the heatedwild-typeprotein (data not shown). Few globular structures wereobservedwhen theDBPs were heated alone,

so it islikely that theaggregates wereformed followingthe

binding oftsDBPs to DNA. Toexaminethepossibility that

the structures

observed

resulted from the spreading

condi-tions,wealso spreadthesamples byusing buffers containing

ammoniumacetate instead of formamide. Underthese

con-ditions,M13 DNAs were collapsed and visualizedastightly

wound structures, whereas thecomplexes formed between

the unheated or heated ts DBPs and M13 DNA showed

structures

resembling

those presented in

Fig.

4B and

C,

respectively (datanotshown). Thus,the structuresresulted

from theexperimentaltreatmentandnottheconditions used

for

spreading.

Effects of metal ions on theDNA-binding activity of DBP.

Mutations in Ad2+NDlts23 and Ad2tslllA are located

withinaputative zinc finger subdomain (37),andtheir effects

mayreflect thefunction of this subdomain. Previous results

(27, 33) and those presented above have shown that the

Ad2+NDlts23 mutation, atthe

permissive

temperature,

re-sults in some decrease in

DNA-binding affinity

without

changing the overall

protein

structure,

indicating

that the

putative

zinc

finger

is

directly

involvedin DNA

binding.

To

investigate this possibility, the

wild-type

and mutantDBPs

were heated at40°C inthe presence ofzinc ionsorEDTA

and their DNA-binding activities were tested by a gel

mo-bilityshift assayusingthe32P-labeled 84-mer oligonucleotide

(Fig. 5). Surprisingly, in the presence of zinc, all of the

mutantproteins retained DNA-bindingactivity at 40°C over

extended periods whereasall ofthe ts DBPs were quickly

inactivated at 40°C in the presence of EDTA. Addition of

zinc to the ts DBPs that had already been heated at 40°C,

however, didnot restoreDNA-bindingactivity. The intrinsic

zinc ion appeared to be bound very tightly in the wild-type

and mutantproteins, because EDTA up to 100 mM had no

effect on the DNA-binding activity ofany ofthe DBPs at

30°C(Fig. 5).

To test whether the stabilizing effect is specific for zinc,

similar experiments were done by usingthe closely related

metalions

Cd2",

Hg2+, and

Co2"

and unrelated ions such as

Mn2"

and

Mg2"

(Fig. 6).Noneoftheseions protectedthe ts

DBPs frominactivationat40°C. Infact, both the wild-type

and mutant proteins lost DNA-binding activity after brief

incubation withCdCl2 at30°CorHgCl2at0°C (Fig.7). The

results imply that cadmium and mercury, owing to their

atomic similarity to zinc, readily substitute for zinc in the

protein, but that these atoms may betoo largeto formthe

exact "finger" structurethatmaybe essential for

function-ing ofthe DBP. The Hg ion also has the ability to modify

sulfhydryl groups in the protein, which may contribute to

inactivation ofthe DBP.

DISCUSSION

This work demonstratesthat thefunctionalchangesin the

Ad2+NDlts23, Ad2tslllA, and Ad5tsl25 ts DBPs at the

nonpermissive temperature can be ascribed to structural

alterations in the

proteins.

The structural alteration was

sufficienttochange the

accessibility

of

proteolytic

enzymes

totheircleavage sites. However, theactualchanges appear

to be minimal-the unheated and heated

proteins

shared many bands whentheyweredigestedwithrelatively

specific

proteases

(trypsin,

chymotrypsin,

and

thermolysin),

and such bands were not obtained with the

totally

denatured proteins.

Our EM studies suggest that the mutationscause

changes

notonly in proteinstructure butalso in the self-association

ofthe DBP. The DBP self-associates intwodifferent

situa-tions: oligomer formation in solution and

cooperative

bind-ingtoDNA.Studiesinotherlaboratories

(29)

have indicated

"i"m

AMIL.- _1:0

VOL.66, 1992

AL,mLA&A&

IW 0

16

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[image:6.612.71.546.73.213.2]

486 TSUJI AND KITCHINGMAN

CdCI2

000

15°0

3000

Inc. time r -

r---C---

00r-(men.)

0 6012018010

2040 60 5 10 15 20

DBP-PDN-D-NSA-S

free DNA

Cd2+

Cd2

F ----I

_ cs

_In

U-- =

Hg

2+

U-)

iSw CD to

3. 4 s

co2+

N3 U-)I

_ rl CNJ

= CM _ : cn en c

a

[email protected]~ 4 e4i,4

HgCI2

6

~000

1500

3000

Inc.

time

,

C 3

(min.)

0 5

10

20

40 5 10 20 40

5

10 15

DBP-DNA-S*

free DNA -

=U

_

I___

FIG. 6. Effects of various metalionsontheDNA-binding

activ-ity of DBPs. DBPs (200 ng)wereincubatedat40°Cfor 30mininthe

presence 1 mM EDTA alone or with 2 mM zinc acetate, CdCI2, HgC12, CoS04,MnCl2,orMgCJ2. Theywerethen mixed with 4ngof

32P-labeled 84-mer DNA andelectrophoresed througha1%agarose

gelat4°C. wt,wildtype.

that the wild-type DBP probably exists as a trimer in solution, although itcanbefound in various otheroligomeric forms, depending on the concentrations ofthe protein and

the salt in solution. Cooperative DNA binding is a charac-teristic commonto many prokaryotic and eukaryotic DBPs

that are involved in DNA replication, including the adeno-virus DBP. Complexes formed between thets DBPs heated at 40°C and M13 DNA contained large, marblelike

aggre-gates.Theseaggregatesmayresult fromanaberrationinthe DBP-DBPinteraction involved in cooperative DNA binding because they appeared to be formed in association with bindingto DNA.

The in vitro studies described here and those previously published (33) demonstrate that the structural and functional changes in thetsDBPsatthenonpermissivetemperatureare irreversible. Thisfinding is incontrast tothose of previous in vivo studies (11, 35), in which viral DNA synthesis in Ad5tsl25-infected cells was blocked at the nonpermissive temperature but this effectwas readily reversed by a shift downtothepermissivetemperature,evenin thepresenceof cycloheximide. Ifaggregates such as those seen here are formed in vivo, theremust bea mechanism for disaggrega-tion ofthe DBP in vivo but notin vitro.

Are the mutations that cause the temperature sensitivity phenotype located in functional or structural amino acid residues?Functional mutations havebeen defined as

muta-tion ofanamino acid residue in the protein causingachange in function without altering the overall protein structure.

Structural mutations are defined as those that affect the stability, and hence function, ofaprotein (36). Our findings

FIG. 7. Inactivation ofwild-typeDBPbycadmiumandmercury.

Wild-type DBP (250 ng)wasincubated for variousperiodsat0, 15,

or30°C in the presence of 1 mMEDTA, 2 mM 2-ME, andeither CdCl2orHgCl2at2 mM. Afterincubation,EDTA anddithiothreitol

wereaddedtothesamplesat4°Ctoafinal concentration of 2 mM

each andDNA-bindingactivitywastestedbythegel mobilityshift

assaywith5ngof32P-labeled84-mer. Inc., incubation.

do notfitprecisely with this categorization. The mutations do not lead to global conformational changes but to more

subtle alterationsresultingin changesin the protease

cleav-age patterns. Previous in vivo studies suggested that the Ad5tsl25 DBP might be degraded at the nonpermissive temperature (11, 34), but our in vitro and EM studies indicate that loss of the epitope recognized by the antibody used for immunoprecipitation in the studies ofGinsberg et al. (11) is a more likely explanation. This loss could be causedby the small conformational changeorby aggregation

of the DBP. The Ad2+NDlts23 DBP is apparently stablein vivo, and this appears to be a functional mutation on the basis of thecriteria cited above.However,wedonotbelieve that the mutations are in amino acid residues directly in-volvedin DNAbinding but think, instead, that both the zinc finger region and the region defined by the Ad5tsl25 muta-tionareimportant for the conformation of the DNA-binding pocket. In support of this possibility, studies by Vos etal. (38) demonstrated that the DBP produced by an in vitro translation systemwasfunctional only when itwas synthe-sized in the presenceof zinc.

Functionalinactivation of thetsDBPs invitro by. heating canbeprevented by prior association with either ssDNAor antibody. Stabilization of the DBP by bindingtossDNAis in agreement with the results ofin vivoexperiments showing thatoneround of DNAreplication canoccurfollowing shift

up to the nonpermissive temperature in Ad5tsl25-infected cells(11, 34). As the DBPsarereleasedfrom the ssDNAby

OOmM EDTA

(300C

30min)

=-< N

*-="cn _)c

3 .tb sn

-No metcl Ions

l =

ca

cw

vx

u!2

-1n-Mg

2

_ Nq N <U)_- n) U)

Mn2+

U) U) U)

<z r) U')

= C\M CM

- vn _)

*s_* * __ * *

Zn2+

Zn2

1 to

N1

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TEMPERATURE-SENSITIVE ADENOVIRUS DNA-BINDING PROTEINS 487

displacement by the elongating second strand, the molecules

becomefunctionally inactivated.

Thepresenceofapotential metal-binding site inthe DBP

was originally suggested by comparative sequence analysis

(37) of the DBPs from distantly related human adenovirus

serotypes. A consensus sequence of the region is

-His-X-Cys-(X)3-Glu-Gly-(X)3-Cys-X-His-, which is similarbut not

identicaltothe typical zinc finger motif(4, 7)found inmany

sequence-specific double-stranded DBPs. Both the Ad2+

NDlts23 and Ad2tslllA mutations are located around the

middle of this sequence, and theamino acid residues

substi-tutedbythese mutationsarehighly conserved among seven

different adenovirus serotypes (37). A key question is

whether this zinc fingerlikeregion is directlyinvolved in the

interaction with DNA or serves as a structural core for

protein folding. Although our results do not clearly

distin-guish betweenthese twopossibilities,we favorthelatter, in

view ofstudies by Vos et al. (38), whichdemonstrated that

the DBP produced by an in vitro translation system was

functional only when it was synthesized in the presence of

zinc.

In summary, we have demonstrated that the

Ad2+ND1

ts23, Ad2tslllA, and Ad5tsl25 ts mutants have an altered

protein structure at the nonpermissive temperature. The

structural alteration is apparently linked to the functional change of these ts DBPs in DNA binding and in DNA

replication. The ts DBPs bound to ssDNA or antibody are

more resistantto inactivation at the nonpermissive

temper-ature than are the free proteins. We have also presented

evidence suggesting potential significance of zinc and the

putative metal-binding subdomain for the structure and/or

function ofthe DBP.

ACKNOWLEDGMENTS

Wethank J.Sublett and P. Stow forhelp with tissueculture, G. Murti for performing the EM studies, and Christy Wright for editorial assistance.

This workwassupported bytheNational Institutes of Health and

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