Association of Holliday-structure Resolving Endonuclease VII with gp20 from the Packaging Machine of...

Association of Holliday-structure ResolvingEndonuclease VII with gp20 from the PackagingMachine of Phage T4

Stefan Golz and BoÈ rries Kemper*

Institut fuÈ r Genetik, d.UniversitaÈt zu KoÈln, ZuÈ lpicherStrasse 47, 50674, KoÈlnGermany

Endonuclease VII (endo VII) is the product of gene 49 (gp49) of bacterio-phage T4. It is a Holliday-structure resolvase (X-solvase) responsible forclearing branched replicative DNA prior to packaging. Consequently,mutations in gene 49 are unable to ®ll heads to completion because unre-solved branches stop translocation of DNA. A likely association of gp49with heads or proheads, however, could not be shown in the past. Wehave investigated whether gp49 could be part of the transientlyassembled packaging machine (the ``packasome'') located at the base ofproheads. Using puri®ed proteins gpl6, gpl7 and gp20, which are con-stituents of the packasome, we found that gp49 binds tightly to gp20 anddoes not bind to gpl6 or gpl7. Quanti®cation revealed that one dimer ofgp49 binds one monomer of gp20. Notably, dimerisation of gp49 was anessential prerequisite for complex formation with gp20, and the dimerisa-tion-de®cient point mutation His-EVII-W87R showed only residual af®-nity to gp20. Furthermore, truncated peptides of gp49 de®cient in dimerformation to various degrees were found to be impaired in binding togp20. In contrast, the cleavage-de®cient mutation EVII-N62D bound nor-mally to gp20. The cruciform DNA (cf-DNA) resolving activity typical ofendo VII is maintained in gp20-gp49 complexes. Furthermore, the com-plexes bind cf-DNA in the absence of Mg2� as demonstrated by electro-mobility shift assays. The binding of the complexes to cf-DNA occurs viagp49, since gp20 alone does not bind cf-DNA. In conclusion, these ®nd-ings are consistent with a model in which gp49 is an integral part of thepackaging machine of phage T4.

# 1999 Academic Press

Keywords: Protein-protein interaction; DNA-protein interaction; DNAcondensation; protein puri®cation; chromosome structure*Corresponding author

Introduction

Maturation of phage T4 occurs late during theinfection cycle by assembly of separately pre-formed structural components like base plates, tail®bres, tails and heads, each processed via indepen-dently operating assembly lines (Edgar & Wood,1966). Packaging of DNA into preformed heads(proheads) is a key event during phage assemblythat precedes the attachment of tails and the ®nal®nishing of infectious particles. The packaging of

DNA requires a packaging enzyme or terminaseand proceeds by a head-full mechanism. The termi-nase of phage T4 is composed of two protein sub-units, gp16 and gp17. The larger subunit, gp17(68 kDa), binds ATP and exhibits a unique exonu-clease activity (Franklin et al., 1998); the smallersubunit, gp16 (18 kDa), is a double-stranded DNA(ds-DNA) binding protein with a strong tendencyto self aggregate in ordered high molecular massstructures (Lin et al., 1997). DNA translocation intoproheads is coupled to ATP hydrolysis. Afterinitiation of packaging, DNA molecules are reeledoff an oversized concatemeric precursor until a``head-full''-signal triggers the terminase to per-form the ®nal cut concluding the packaging event(Streisinger, 1966).

In phage T4, the start site for packaging is notrestricted to de®ned sequences in the genome as

E-mail address of the corresponding author:[email protected].

Abbreviations used: endo, endonuclease; cf-,cruciform; ss-, single-stranded; ds-, double-stranded;BSA, bovine serum albumin; EMSA, electromobilityshift assay.

Article No. jmbi.1998.2398 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 285, 1131±1144

0022-2836/99/031131±14 $30.00/0 # 1999 Academic Press

has been shown for other large phages, forexample the cos site in phage l (for a review, seeMurialdo, 1991) or the pac site in phage T7 (for areview, see Black, 1989). This explains why thegenetic map of T4 is circular permuted, contrastingsharply with a physically linear organisation of thegenome (MacHattie et al., 1967).

The DNA packaging machinery is located at oneof the two small-side vertices of the prohead. It isthe same vertex that is assembled ®rst on a dode-cameric ring-structured base made from portal pro-tein gp20 that itself is anchored in the membranevia contact to gp40 (Brown & Eiserling, 1979; Hsiao& Black, 1977).

Genetic evidence suggested that the terminaseprotein gp17 is associated with portal proteingp20. This was concluded from identi®cation ofsecond site suppressor mutations in gene 17 com-pensating cold sensitive mutations (cs) in gene 20for their de®ciency in DNA packaging despite for-mation of functional proheads (Hsiao & Black,1977). gp16 was recently shown to bind to gp17(Rao & Black, 1988), suggesting a layered proteinarchitecture of the packaging machinery: head-gp20-gp17-gp16.

An early screen for mutations in phage T4 led tothe discovery of gene 49, which was classi®ed atthat time by the phenotype of 49ÿ mutants as amorphopoietic gene required for packaging DNAinto preformed phage heads (Epstein et al., 1964).Further studies revealed, however, that the mor-phopoietic phenotype of 49ÿ mutants is due to adefective endonuclease (endo VII) that is respon-sible for the removal of branches from precursorDNA prior to packaging (Frankel et al., 1971;Kemper & Garabett, 1981). In the absence of activegp49, packaging proceeds until branches in theDNA, originating from earlier occurring recombi-nation and replication events, approach theentrance of the head, blocking the translocationprocess by sterical hindrance (Frankel et al., 1971;Kemper & Brown, 1976).

Detailed analyses revealed that endo VII cleavesbranched DNAs like Holliday structures (Mizuuchiet al., 1982) and Y-junctions (Jensch & Kemper,1986) and a broad range of other DNA substrateswith an altered secondary structure. These sub-strates are single-strand overhangs, ¯aps, nicks,gaps, mismatching nucleotides, heteroduplexloops, bulges, bulky adducts, curved DNA(Kemper, 1997) and apyrimidinic sites (unpub-lished results). The enzyme introduces time-delayed staggered nicks two to six nucleotides 30¯anking the target. Nicking at mismatching basescan trigger excision repair events performed byDNA polymerase and DNA ligase in vitro(Birkenkamp & Kemper, 1995; Solaro et al., 1993)and in vivo (Grebenshchikova et al., 1994). Theseresults suggest that endo VII plays an importantrole throughout the whole life-cycle of phage T4and resolution of branches prior to packaging maynot be the only function of the enzyme (Kemper,1997).

The broad substrate pro®le of endo VII suggeststhat the enzyme has ample opportunities in vivo tocleave DNA. While on one hand these cleavagesinitiate repair, on the other hand they threaten thestability of the genetic material. In fact, clonedendo VII was found to be highly toxic for cells.The basal level of uninduced tight repression plas-mid systems degrades the genome very quickly(Kosak & Kemper, 1990). Surprisingly, endo VII isnot toxic for T4 itself, suggesting the existence ofcontrol mechanisms for protection of its ownDNA. Physical localisation of the enzyme to sitesof action was proposed as a possibility for exertionof such control. As a guarding role in DNA packa-ging, the ``packasome'' located at the entrance ofthe head could provide a solid base for positioningan aggressive nuclease like endo VII. The ideaprompted us to investigate the af®nity of endo VIIfor known constitutents of the packasome, whichare to date the portal protein gp20 and the termi-nase proteins gp16 and gp17. We report here thatendo VII indeed binds as a dimer to gp20 but notto gp16 or gp170, which is a truncated but packa-ging active smaller version of full-size gp17 (fordetails, see below). gp170 in turn, however, wasshown to bind directly to gp16 and gp20. The ®nd-ings are discussed in favour of a model in whichthe packaging machinery is built from a centralplatform of gp20 at the base of the prohead, pro-viding suf®cient space for binding gp49 and gp17with associated gp16.

Results

Small amounts of endo VII leaking from unin-duced, overexpressing plasmids are highly toxicfor cells and quickly destroy their DNA (Kosak &Kemper, 1990). The same enzyme, however, is tol-erated in T4-infected cells and its activity is evenrequired for normal growth, in particular forpackaging the phage DNA (Epstein et al., 1964). Itwas therefore reasoned that T4 should possessmechanisms to protect its own DNA againstuncontrolled damage by endo VII. Incorporationand concentration of the enzyme in proteinmachines that are localised to their sites of action(Alberts, 1998) was envisaged as a way to keepgp49 away from free DNA. This idea prompted usto study protein-protein interaction between gp49and puri®ed proteins gp16, gp17 and gp20, whichwere described as constituents of the DNA packa-ging machine, the packasome (Black, 1995).

Purification of proteins gp16, gp170 and gp20

T4 genes 16 and 20 were cloned as PCR productsinto vector pET11a for protein expression. Gene 16was cloned into the unique NdeI restriction site;gene 20 between the restriction sites XbaI andBamHI of the same vector. Plasmid pJF17, kindlygiven to us by G. Mosig, was used for expressionof gp17 (Franklin & Mosig, 1996). The proteinswere overexpressed in Escherichia coli BL21(DE3)

1132 Holliday-structure Resolving Endo VII

after induction with IPTG and puri®ed from crudeextracts using the puri®cation protocols describedin Materials and Methods. As mentioned there, the®nal product comprised a truncated version ofgp17, protein gp170, with a molecular mass of59 kDa instead of 68 kDa for the full-size proteingp17. Since gp170 was shown to be active in anin vitro packaging assay (see next paragraph), theshortened version of the protein was used through-out these studies. The purity of the proteins gp16,gp170 and gp20 was greater than 99.99 % as deter-mined on silver-stained, SDS-containing polyacryl-amide gels (Figure 1, lanes 2, 3 and 5).

During puri®cation of gp16 and gp170, function-ality of the proteins was monitored by the in vitropackaging assay as described (Rao & Black, 1988).Addition of the puri®ed proteins to reactions inextracts lacking one of the respective proteins com-plemented the in vitro packaging system to fullfunctionality (results not shown). The functionalityof puri®ed gp20 was not directly testable. The abil-ity of the protein to form high molecular massaggregates of >700 kDa was taken as an indicatorfor the potency of the protein to form complex por-tal base structures (Driedonks, 1981).

Protein-protein interactions studied byaffinity chromatography

For initial studies of protein-protein interactionsbetween endo VII and the packaging proteins, af®-nity chromatography with immobilised endo VIIwas performed. Solutions containing proteinsgp20, gp16 and gp170 were passed through anendo VII-Sepharose af®nity column prepared andprocessed as described in Materials and Methods.Bound proteins were eluted with a linear salt gra-dient of KCl in the same buffer, and the proteincontent of interesting fractions was analysed bySDS-PAGE. Columns containing immobilisedbovine serum albumin or NHS-Sepharose inacti-vated with ethanolamine were used for controls.Protein samples were applied in small volumes tothe column and allowed to remain undisturbedthere for 15 minutes to ensure equilibrium bindingbefore the elution was started.

Typical elution pro®les obtained in runs withproteins gp16, gp170 and gp20 are shown inFigure 2. Neither gp16 nor gp170 was retained onthe column, and the entire protein samples wererecovered from the ¯ow-through. In contrast,

about 70 % of the input of gp20 was bound to thecolumn and eluted at 265 mM KCl, while theremaining 30 % of the input appeared in the ¯ow-through.

The association constant for the gp20-gp49 com-plex was calculated from these numbers as1.27 � 10ÿ7 M. This was based on experimentsusing 50 mg of gp20 in the mobile phase and 1 mgof gp49 immobilised in the column: 35 mg (70 % ofthe input) of gp20 was complexed with gp49{.

Protein-protein interaction studied bygel filtration

Further studies of af®nities between proteinsgp16, gp170, gp20 and gp49 were performed by gel®ltration using calibrated FPLC-Superose columns.Pairs of proteins were mixed in all possible combi-nations, preincubated at 16 �C and then passedthrough the molecular sieve as described inMaterials and Methods. After the run, sampleswere taken from each fraction, precipitated withtrichloracetic acid, and their protein content deter-mined by SDS-PAGE. A typical chromatogram isshown in Figure 3(a) for an equimolar mixture ofproteins gp16 and gp170 using Superose 12 for gel®ltration. Monomers of gp16 with a molecularmass of 18 kDa have a strong tendency to formhigh molecular mass complexes of about 150 to200 kDa (Lin et al., 1997). These complexes elute in

Figure 1. PAGE analyses of puri®ed proteins. Samplesof 500 ng of puri®ed proteins gp16 (lane 2), gp49 (lane3), gp170 (lane 4) or gp20 (lane 5) were separated on anSDS/15 % polyacrylamide gel and silver-stained forvisualisation. Marker (M) proteins (lane 1) are indicatedby their molecular mass (kDa) to the left of the Figure.

{ The association constant was calculated by theequation K � [ML]/[M][L], in which [ML] is theamount of gp20-gp49 complexes formed at anequilibrium during the preincubation period, [M] isthe amount of immobilised gp49 in the column, [L] isthe amount of gp20 that does not bind to gp49. Itfollows:

K � �2:2� 10ÿ13��5:5� 10ÿ8��3:17� 10ÿ13� � 1:27� 10ÿ7 M

Holliday-structure Resolving Endo VII 1133

the ®rst peak around fraction 18, labelled gp16 inFigure 3(a). gp170 with a monomeric molecularmass of 59 kDa, which does not self-aggregate(Franklin & Mosig, 1996), elutes around fraction41. Complexes gp16-gp170 elute in front of thegp16 peak giving a distinct shoulder (marked witha grey bar in Figure 3(a); protein gel in Figure 3(b),lanes 2 and 3).

In conclusion, these analyses con®rmed earlierresults obtained on the interactions between theseproteins. We found in particular that (i) gp170binds gp16 (Figure 3b, lanes 2 and 3; Black, 1995),(ii) gp20 binds gp170 (Figure 3(b), lanes 7 and 8)and (iii) gp170 binds both gp20 and gp16(Figure 3(b), lanes 21 and 22). No complex wasformed between gp20 and gp16 (Figure 3(b), lanes4 to 6), suggesting a layered arrangement of thethree proteins in the packasome with gp170 sand-wiched between gp20 and gp16.

Combinations of gp49 and gp16 (Figure 3(b),lanes 9 to 11) and gp49 and gp170 (Figure 3(b),lanes 15 to 17) did not form complexes, indicatingthat endo VII does not contact the terminase pro-teins. However, gp49 forms complexes with gp20that are formed in the absence of gp16 or gp170(Figure 3(b), lanes 12 and 13) as well as in theirpresence (Figure 3(b), lanes 23 and 24). Asexpected, in a mixture containing all four proteins,four-protein complexes containing gp49-gp20-

Figure 3. Gel ®ltration analyses. Samples containingcombinations of equal amounts of proteins in variouscombinations of gp16, gp170, gp20 and gp49 werepassed through an FPLC Superose2 column asdescribed in Materials and Methods. The protein contentof each fraction collected was determined on SDS/15 %polyacrylamide gels silver-stained for visualisation.(a) Elution pro®le of the mixture gp16 and gp170. Theprotein content was monitored by measuring absor-bance at 280 nm (ÐÐ). Two main peaks containinggp16 or gp170 are labelled accordingly in the Figure.The shoulder eluting ahead of gp16 contains complexedgp16 � gp170. A grey bar denotes the fractions used fordetermining their protein content on SDS/polyacryl-amide gels in (b). The column was equilibrated with sixmarker proteins. Two positions marked with arrows inthe Figure denote BSA (66 kDa) and alcohol dehydro-genase (150 kDa). (b) Two SDS/polyacrylamide gels areshown containing key fractions from separate gel ®l-tration experiments. Fractions containing single proteins(Es) or complexed proteins (Ec) after chromatography ofthe respective protein mixtures (I) are indicated abovethe Figure. Positions of each protein on the gel are indi-cated to the right of the gel image.

Figure 2. Af®nity chromatography. Samples contain-ing 20 mg of (a) gp16, (b) gp170 or (c) gp20 were loadedonto an endo VII af®nity column and processed asdescribed in Materials and Methods. Elution of each ofthe proteins with a linear KCl gradient ( � � � � � ) was mon-itored by measuring the absorbance at 280 nm (ÐÐ).


gp170-gp16 were obtained (Figure 3(b), lanes 23and 24), indicating that endo VII is part of thepackasome.

Further attempts to resolve the complexes byglycerol gradient centrifugations gave basically thesame results. Complexes of >700 kDa were foundin mixtures of gp20 with gp49. The resolution ofproducts in the gradients, however, was less goodthan that obtained by molecular sieving and gp20homo-complexes could not be distinguished fromgp20-gp49 hetero-complexes (data not shown).

Quantification of the components in gp20-gp49 complexes

To further investigate the content of the singleproteins in the gp20-p49 complexes, quantitativebinding experiments were performed usingimmobilised gp20. A distinct amount of gp20 wasallowed to bind to the inner surface of the wells ofmicrotiter plates. After exhaustive washings toremove residual free gp20, followed by a blockingreaction to silence unspeci®c binding sites on theplastic surface, an excess of gp49 was added toeach well. After a suf®cient incubation period, sur-plus gp49 was removed by repeated washings.

Complexed proteins were then removed fromthe wells by washing with 10 % (w/v) SDS. Thesamples were analysed by PAGE and the amountof each component was determined by comparingthe respective bands on silver-stained SDS/poly-acrylamide gels with standards of each protein.The results revealed that a maximum of 150 ng ofgp49 became bound by 250 ng of gp20 (Figure 4(a),lane4). Taking the molecular mass of one monomerof gp20 (60.5 kDa) and one monomer of gp49(18 kDa), we calculated 3.9 � 10ÿ9 mol of gp20binding 8.3 � 10ÿ9 mol of gp49. This is approxi-mately twice the amount of gp49 than gp20,suggesting that one dimer of gp49 was bound byone monomer of gp20 (Figure 4(a)).

Effect of protein-protein interaction onendo VII activity

Next we wanted to know whether the nucleo-lytic activity of endo VII would be affected by theaddition of packaging proteins. Standard endo VIIcleavage reactions were performed in the presenceand in the absence of proteins gp16, gp170 andgp20 using cruciform DNA (cf-DNA) CF01 as sub-strate with 50end-labelled strand 4 (Golz et al.,1995). Typically, endo VII introduced one majorcut and one adjacent minor cut 30 ¯anking the junc-tion (Figure 5, lanes 1 and 2; marked CS). Proteinsgp16, gp170 and gp20 did not cleave cf-DNA(Figure 5, lanes 3 and 13 to 18). When these pro-teins were included in reactions with gp49, therelative cleavage ef®ciency of endo VII was mark-edly reduced in the presence of gp20 and reductionof activity became clearly visible with 50 ng ofgp20 (Figure 5, lanes 4 to 6). The nucleolyticactivity of endo VII remained completely unaf-

fected after additions of gp16 or gp170, added sin-gly or in combination (Figure 5, lanes 19 to 23).Neither was the apparent reduction of activityexerted by gp20 on endo VII cleavage released byfurther additions of gp16 and gp170, and endo VIIremained low in activity in the presence of gp20(Figure 5, lanes 24 to 26).

When gp20 was preincubated with gp49 in thecomplete reaction mixture without cf-DNA and thecleavage reaction was started later with addition ofthe DNA, slightly enhanced reduction of the nucle-ase activity was observed (Figure 5, compare lanes4 to 6 with lanes 7 to 9). Preincubation of gp20 incomplete reaction mixtures with cf-DNA followedlater by addition of gp49 gave the same reductionof the cleavage reaction as seen without preincu-bation (Figure 5, compare lanes 4 to 6 with lanes10 to 12). These results suggest that the cleavage

Figure 4. Quanti®cation of gp20-associated gp49.Wells of microtiter plates were seeded with gp20 andprocessed as described in Materials and Methods. gp49was allowed to bind to immobilised gp20. (a) Total pro-tein was removed from the wells by washing with SDSand analysed by denaturing PAGE. Lane 1, marker pro-teins (M) are indicated by their respective molecularmass (kDa) on the left of the Figure. Lane 2, control ofresidual binding of gp49 (49) to milk powder (mp)blocked wells. Note that the protein bands on the gelderive from milk powder. Lane 3, quanti®cation of thetotal amount of gp20 bound to one well ®nalised byblocking with milk powder. Lane 4, quanti®cation of thetotal amount of gp49 bound to gp20 immobilised andmilk powder blocked in one well. Lane 5, demonstrationand quanti®cation of binding of gp49 to one well with-out milk powder blockage. Positions of proteins gp20(20) and gp49 (49) in the gel are indicated to the right ofthe Figure. (b) Cleavage of radioactively labelled sub-strate CF01 visualised on 10 % polyacrylamide gels.Lane 1, control showing cleavage of strand four of CF01(S) in solution by free endo VII in standard reactionsdescribed in Materials and Methods. Fragments (CS)derived from cleavage at major cleavage sites aremarked to the right of the Figure. Lane 2, mock incu-bated control substrate. Lane 3, residual endo VIIactivity determined in one well that was exposed togp49 after the blocking reaction with milk powder (com-pare (a), lane 2). Lane 4, residual endo VII activity deter-mined in the ¯uid of the last washing step in anexperiment as described for (a), lane 4. Lane 5, endo VIIactivity determined in one well with gp49 bound toimmobilised gp20 as described for (a), lane 4.


reaction is faster than the assembly of the gp20-p49complex that causes the reduction in activity.

Nuclease activity of endo VII is conserved ingp20-gp49 complexes

If gp49 were part of the packasome in vivo, assuggested by the in vitro experiments describedabove, one should expect that its nucleolyticactivity is retained in the complexes in order toperform its resolvase function during DNA packa-ging. This was investigated using immobilisedgp20-gp49 complexes in wells of microtiter platesand cf-DNA as substrate. The protein complexeswere generated and washed as described in theearlier paragraph. Radioactively labelled cf-DNAwas then added to selected wells in endo VII reac-tion buffer containing Mg2�. After incubating theplates for 15 minutes at 37 �C the cf-DNA was ana-lysed on denaturing polyacrylamide gels. Asshown in Figure 4(b) gp49 could still cleave theDNA while bound to gp20 (Figure 4(b), lane 5).The ®nal wash contained low residual cleavageactivity (Figure 4(b), lane 4), while the bound pro-tein showed substantial cleavage activity. Thisindicates that the above described apparent ``inhi-bition'' of endo VII by gp20 is not due to silencingendo VII activity but rather the consequence oflowering the amount of freely accessible gp49 insolution by trapping the enzyme in large gp20-

gp49 complexes. endo VII still retains its enzymaticactivity in gp20-gp49 complexes.

Protein-DNA interaction studied byelectrophoretic mobility shift assays (EMSA)

In order to investigate the DNA-binding abilityof terminase proteins gp16 and gp170 with portalprotein gp20 and nuclease gp49, shift experimentswere performed using single-stranded DNA (ss-DNA), double-stranded DNA (ds-DNA) or cf-DNA as substrates. The shift assays were done asdescribed in Material and Methods. The assayscontained the proteins singly or in combinations asindicated below. Proteins gp32 and UvsY wereincluded for control as ss-DNA and ds-DNA bind-ing proteins, respectively.

While gp32 shifted ss-DNA ef®ciently(Figure 6(a), lane 2; shift position b) none of theproteins from the packasome, gp16, gp170 or gp20,bound ss-DNA under the same conditions(Figure 6(a), lanes 1, 3, 5 and 7). When the shiftexperiments with ss-DNA and gp16, gp170 andgp20 were repeated in the presence of gp32, asupershift was observed with gp170 (Figure 6(a),lane 6, shift position c). This was expected, sincegp170 binds to gp32 (Mosig et al., 1981).

The ds-DNA was not shifted by any of thepackaging proteins either singly or in combi-nations, as indicated in Figure 6(b), lanes 1 and 3

Figure 5. Effect of gp16, gp170 and gp20 on endo VII activity. endo VII activity was determined under standardassay conditions using cruciform DNA CF01 as substrate (labelled strand 4) as described in Materials and Methods.Normally, 100 units of endo VII was added to each reaction and distinct amounts (ng) of proteins gp16, gp170 andgp20 as indicated above the Figure. Prot-pre, samples in which proteins were preincubated for 15 minutes at 16 �C.DNA-pre, samples in which gp20 was preincubated with DNA for 15 minutes at 16 �C. S, uncleaved input DNA. CS,cleaved product.


to 10. Only the control protein UvsY bound ds-DNA successfully (Figure 6(b), lane 2; shift positionb). Neither was cf-DNA shifted by either gp16,gp170 or gp20 (Figure 6(c), lanes 1, 3, 5 and 7).However, gp49 bound cf-DNA successfully(Figure 6(c), lane 2; shift position b) as reportedpreviously (Golz et al., 1997). When pairwise com-

binations of packaging proteins and gp49 wereused, combination gp20 � gp49 gave a supershift(Figure 6(c), lane 8; shift position c), re¯ectingthe above-described interaction between the twoproteins. Combinations gp16 � gp49 and gp170 �gp49 did not supershift the DNA (Figure 6(c),lanes 4 and 6).

In summary, these results indicate that none ofthe proteins previously described as constituents ofthe packasome, singly or in combination, bindsdirectly to DNA in vitro under the conditions usedhere. It is worth noting that the binding of gp49 toDNA is not impaired by the addition of gp20.

Dimerization of gp49 is a prerequisite forbinding gp20

Measurements of the mass ratio of the com-ponents in gp49-gp20 complexes had revealed onedimer of gp49 per monomer of gp20. For localisingthe protein-protein binding domain of gp49, trun-cated peptides were made and studied in bindingreactions with gp20 immobilised in wells of micro-titer plates. Bound endo VII peptides were quanti-tated by exhaustive reactions with polyclonalrabbit anti-endo VII antiserum followed by goatanti-rabbit antibodies coupled to alkaline phospha-tase. Phosphatase activity was determined enzy-matically and used as an indirect measure for theamount of bound antigen. Variation in the ef®-ciency of the recognition of endo VII peptides bypolyclonal antibodies was taken into account.

As summarised in Figure 7, ef®cient bindingwas found with intact protein gp49 containing 157amino acid residues (EVII-wt). The complete pro-tein with a point mutation in codon 62 (EVII-N62D), which completely eliminates the cleavageactivity of endo VII (Golz et al., 1997), shows thesame binding capacity as the wild-type protein(EVII-wt). In contrast, the complete protein with apoint mutation in codon 87 (His-EVII-W87R),which reduces the dimerization ability of endo VIIto below 5 % (Birkenbihl & Kemper, 1998), shows aresidual binding capacity of about 25 %. The possi-bility that the His-tag of the protein His-EVII-W87R is responsible for the effect was excluded bydemonstrating that the dimerization-pro®cient pro-tein His-EVII-H43R carrying the same His-tagshows full binding capacity.

This was further substantiated with a series oftruncated peptides obtained from gp49. Stepwiseremoval of residues from the N terminus, the Cterminus or from both termini resulted in a gra-dual loss of the binding capacity. For example,removal of 10, 20, 64 or 113 residues from the Nterminus reduced the respective relative bindingef®ciencies to 90 %, 90 %, 30 % and 20 % comparedto wild-type. Removal of 7, 10, 55 or 72 residuesfrom the C terminus reduced the respective relativebinding ef®ciencies to 95 %, 70 %, 35 % and 20 %compared to wild-type. An internal peptide reach-ing from residues 78-112 had a relative bindingef®ciency of 30 %. Reduction in relative binding

Figure 6. Electrophoretic mobility shift assays(EMSA). Standard reactions were performed asdescribed in Materials and Methods. Proteins used sin-gly or in combinations were gp16 (16), gp170 (17), gp20(20), gp32 (32), gp49 (49) and UvsY (Y). Distinct shiftpositions are labelled a, b and c at the left side of theFigure. (a) Samples contained 2 fmol of ss-DNA and100 ng of each protein indicated in the Figure.(b) Samples contained 2 fmol of ds-DNA and 100 ng ofeach protein indicated in the Figure. (c) Samples con-tained 2 fmol of cf-DNA CF01 and 100 ng of each pro-tein indicated in the Figure, except for gp49, whichcontained 5 ng.


ef®ciencies between gp49 peptides and gp20 is par-alleled by reduction in dimerisation ef®ciencies.For example, it was recently shown (Birkenbihl &Kemper, 1998) that N-terminal truncated peptidesEVIIpep(21-157) and EVIIpep(55-157) showednearly 100 % dimerization ability, while shorterpeptides EVIIpep(63-157) and EVIIpep(76-157)were not able to dimerise. C-terminal truncatedpeptides EVIIpep(1-120), EVIIpep(1-105) and EVII-pep(1-90) showed about 90 %, 70 % and 410 %dimerisation, respectively. The results presented inthis study suggest strongly that dimerization ofendo VII is a prerequisite for binding to gp20.

Discussion

For the reasons outlined in the Introduction, weinvestigated the af®nity of the Holliday-structureresolving endonuclease VII (gp49) for packagingproteins gp16, gp170 and gp20. The results showthat gp49 binds to gp20 with considerable strength.Binding to gp16 or gp170 was not observed.

The binding of gp49 to gp20 was studied usingdifferent experimental approaches. These includedaf®nity measurements between (i) mobile gp20 andimmobilised gp49 in Sepharose columns, (ii)mobile gp49 and immobilised gp20 in wells ofmicrotiter plates and (iii) both proteins mobile insolution. An association constant of 1.27 � 10ÿ7Mwas calculated by measuring the amount of gp20binding to an excess of immobilised gp49 underequilibrium binding conditions. The value is typi-cal for moderate binding strength. Low bindingconstants are found in enzyme-substrate inter-

actions, e.g. triosephosphate isomerase and glycer-alaldehyde 3-phosphate with 4.7 � 10ÿ4M . Highbinding constants of 410ÿ10M are found in anti-gen-antibody interactions. An extremely high bind-ing constant of 10ÿ15M was measured betweenavidin and biotin. The latter complexes can hardlybe separated under native conditions.

The moderate binding between gp49 and gp20may be advantageous and typical for a dynamiccomplex, permitting limited turnover of com-ponents in general and release of gp49 in particu-lar, ensuring that endo VII can visit other locationsin the cell where its activity is required, e.g. forongoing DNA repair and recombination events(Grebenshchikova et al., 1994; Solaro et al., 1993). Amoderate association constant would also facilitateremoval of gp49 from heads after packaging hadceased; gp49 is not part of matured phage (Deweyet al., 1974).

The packasome may indeed be considered as theplace best suited for a ®nal checkup of the integrityof incoming DNA, with gp49 scanning the DNAfor correctable de®ciencies just prior to transloca-tion into the prohead. This view was supported byan early observation that after release of a tempera-ture block in infections with a temperature sensi-tive (ts) gene 49 mutant (C9ts) a burst of repairsynthesis preceded resumption of packaging. Therepaired DNA was the ®rst portion appearing inthe revived heads after the shift (Laemmli et al.,1974). This was expected, since in 49ÿ infectionspackaging is interrupted physically by DNAbranches obstructing the translocation process infront of the heads. Resolution and repair of thesebranches were expected to occur ®rst after restor-

Figure 7. Binding of gp49 peptides to gp20 quanti®ed by ELISA. The binding of gp49 wild-type, mutant proteinsand peptides to gp20 was determined and quanti®ed using sandwich ELISA technology as described in Materialsand Methods. Proteins and peptides used in the study are designated to the left of the Figure with combinations ofletters EVIIpep and numbers in parentheses denoting the amino acids present in the peptide. The relative size of thepeptide and its origin in the protein is indicated with black lines. Pre®x His indicates His-tagged proteins and pep-tides. Mutations are marked by crosses on the lines. Horizontal black columns indicate the measured relative amountof the respective proteins or peptides that bind to immobilised gp20. Sizing of the bars is related to 100 % binding byEVII-wt.


ing permissive conditions (Kemper & Brown,1976).

Proteins gp16 and gp170 were not bound bygp49. Both proteins are, however, constituents ofthe terminase that is responsible for initiating DNApackaging and later for cutting the DNA from theprecursor concatenate. Although gp16 is not essen-tial and situations are known where packagingproceeds in its absence in vivo as well as in vitro, itmodulates the activity of gp17 and improvespackaging rates (Black et al., 1994; Rao & Black,1988). Binding between gp16 and gp170 wasdemonstrated here by gel ®ltration analyses sup-porting the results of others (Franklin et al., 1998;Lin et al., 1997). While binding between gp16 andgp20 was not observed, binding between gp170and gp20 did occur. Taken together these ®ndingssuggest a sandwich-like orientation of the threeproteins in the packasome with the portal proteingp20 building the base at the entrance of the headand binding gp170, which in turn binds gp16 (Rao &Black, 1988). This was supported here by exper-iments demonstrating the formation of stable three-protein complexes in samples with the three proteinsgp16, gp170 and gp20 (Figure 3(b)). Furthermore, theaddition of gp49 as a fourth protein in the mixtureled to formation of four-protein complexes, indicat-ing that there is free space in the packasome avail-able for gp49. Quanti®cation of the results indicatedthat the terminase proteins and gp49 did not com-pete for binding, consistent with a view of a multi-protein machine in which different components areassembled for concerted action (Figure 3(b)). Dem-onstration of the persistence of the nuclease activityof gp49 after binding in gp49-gp20 complexes is alsoin agreement with the model.

Only 10-15 % of the incubated proteins gp49 andgp20 formed stable complexes that were separtableby gel ®ltration. For example, gp16 and gp170delivered only about 10 % of the input protein inthe form of stable gp16-gp170 complexes. This is inagreement with the previously described low stab-ility of the gp16-gp17 complex (Rao & Black, 1988).

Determination of the stoichiometry of proteins ingp49-gp20 complexes revealed one dimer of gp49per monomer of gp20. Dimerisation was found tobe a prerequisite for the protein-protein contact,since a dimerisation-de®cient mutation in endo VII(His-EVII-W87R) resulted in a protein largelyde®cient in binding gp20. The portion of the pro-tein of endo VII responsible for binding gp20 wasfurther analysed by using truncated peptides ofendo VII in ELISA assays. The loss of the dimerisa-tion ability of the peptides paralleled the loss of theirbinding ability with gp49, strengthening the viewthat the dimer is indeed the smallest unit of gp49 uti-lised for protein binding. Provided that gp20assumes the suggested dodecameric toroidal proteinstructure at the base of the prohead, there would bespace suf®cient for 12 dimeric units of gp49.

Which proteins of the packasome bind DNA?We approached the question by studying bindingof proteins gp16, gp170, gp20 and gp49, alone or in

combination, to model DNA substrates made fromshort synthetic oligonucleotides. As shown hereand demonstrated earlier, gp49 binds strongly tocf-DNA (Golz et al., 1997; and see Figure 7(a)).With the exception of gp170, none of the other pro-teins bound any of the DNAs. Protein gp170 doesbind to ss-oligonucleotides in the presence of ss-DNA binding protein gp32 (Figure 7(a)). The bind-ing re¯ects an earlier observed af®nity betweengp17 and gp32, and the observed retardation inthe gel is most likely caused by protein-proteinrather than protein-DNA interaction (Mosig et al.,1992). In contrast to these results, gp17 wasrecently described to bind ss-DNA. As a matter offact, af®nity chromatography over ss-DNA/agarosewas used there for successful puri®cation of theprotein (Franklin et al., 1998). One likely reasonwhy gp17 was found to bind to ss-DNA in thoseexperiments and not to ss-oligonucleotides in ourexperiments might stem from the fact that theauthors had used high molecular mass DNA forpreparation of ss-DNA/agarose, which in fact stillhas a high content of ds-DNA and secondary struc-tures due to spontaneous internal snap-back fold-ing (Marvin & Schaller, 1966; Schaller et al., 1969).Furthermore, gp17 binds preferentially to junctionsbetween ss-DNA and ds-DNA, while blunt-endedds-DNA was not bound (Franklin et al., 1998).

gp16 promotes ampli®cation of gene 17 by site-speci®c recombination using short sequences (Wu& Black, 1995). gp16, which is strongly self-associ-ating (Lin et al., 1997), performs synapsis-promotedrecombination between DNA sequences repeatedmany times along the oversized DNA moleculesmade during replication (Wu & Black, 1995). Themolecular mechanisms of these events and theirrelation to DNA packaging are still unknown. It istempting to speculate, however, that the Holliday-structure resolving activity of gp49 is required forthese DNA exchange reactions, adding anotherplausible reason why endo VII should be prefer-ably located at the base of heads. The ®nding thatthe initiation of DNA packaging is not impaired in49ÿ infections (Luftig et al., 1971) suggests, indeed,either that synapsis-promoted recombination doesnot depend on active gp49 at this early stage ofpackaging or that this site-speci®c recombination isnot essential for DNA packaging.

The described af®nity between portal proteingp20 and gp49 is also seen here, in support of theidea that sensitive sites in the cell can, in principle,be protected from attacks by hazardous enzymesby their integration into protein machines, therebylowering their free concentrations in solution. Theclose proximity of proteins gp20, gp49, gp16 andgp17 at the base of the prohead indicates intimatefunctional interdependence and suggests that they,together with other components, like, for example,DNA polymerases and DNA ligases, which arerequired for repair, are all part of the samemachine. As expected, endo VII was also found tobind to puri®ed heads from 16ÿ, 17ÿ and 49ÿ infec-tions in vitro. Experiments to further localise the


binding sites of gp49 on the surface of the headsare under way.

From this model one may predict mutations toexist in gene 20 or gene 49 that eliminate bindingbetween the two proteins. Furthermore, second sitesuppressor mutations mapping in either of therespective non-mutated genes that restore the bind-ing should exist. These predictions will be tested.

Materials and Methods

Plasmids, bacteria and phage

Expression vector pET11a and E. coli strain BL21(DE3)were obtained from W.F. Studier (Studier et al., 1991).E. coli strains DH5a, CR63 and B/S were from our straincollection. DH5a was used for sequence analyses(Hanahan, 1983). Phage T4 strains amN66 (16ÿ), amN56(17ÿ) and amB17 (23ÿ) were from our strain collection.Plasmid pJK17 was kindly provided by G. Mosig (Nash-ville, USA; Franklin & Mosig, 1996).

Growth of bacterial cultures

E. coli BL21(DE3) harbouring plasmids pSG20 (gene20), pSG16 (gene 16) or pJK17 (gene 17) were grown in afermentor in LB medium with 100 mg/ml ampicillin at37 �C under stirring (700 rpm) and heavy aeration(6 l/minute) to an A578 of 0.8. Growing cells wereinduced by the addition of IPTG to a ®nal concentrationof 1 mM for 1.5 hours. Cells were harvested by centrifu-gation in a Sorvall centrifuge at 3000 g for 15 minutes at4 �C. The pellet was washed twice with ice-cold l-bufferand then frozen in liquid nitrogen and stored at ÿ80 �C.

Buffers and media

Buffer A contained 10 mM Tris-HCl (pH 8.0), 10 mMMgCl2, 1 mM EDTA, 2 mM PMSF, 10 % (v/v) glyceroland 10 mM 2-mercaptoethanol. Buffer AU contained10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM PMSF,10 % (v/v) glycerol, 10 mM 2-mercaptoethanol and 4 Murea. endo VII reaction mix (10�) contained 500 mMTris-HCl (pH 8.0), 100 mM MgCl2 and 100 mM 2-mer-captoethanol. Buffer B contained 10 mM Tris-HCl(pH 8.0), 1 mM EDTA, 10 % (v/v) glycerol. Buffer C con-tained 10 mM KHPO4/KH2PO4 (pH 6.0), 1 mM 2-mer-captoethanol, 1 mM EDTA and 10 % (v/v) glycerol.Buffer BATP contained 10 mM Tris-HCl (pH 8.0), 1 mMEDTA, 1 mM 2-mercaptoethanol and 1 mM ATP. M&Gstopmix (denaturing loading buffer) contained 90 %(v/v) formamide, 10 % (v/v) TBE and 0.1 % (w/v)bromophenol blue. LB-medium contained 5 g/l NaCl,5 g/l yeast extract and 10 g/l tryptone. NE-buffer(10 � native electrophoresis buffer) contained 67 mMTris-HCl (pH 8.1), 33 mM sodium acetate and 20 mMEDTA. Loading buffer (5 � LB-buffer) contained 40 mMTris-HCl (pH 7.5), 4 mM EDTA, 25 % (v/v) glycerol,400 mg/ml BSA and 0.1 % (w/v) bromophenol blue.TEDB-buffer (EMSA binding buffer) contained 20 mMTris-acetate (pH 7.4), 5 mM EDTA, 1 mM DTT and(1 mg/ml BSA). TBE-buffer contained 45 mM Tris-borate(pH 8.0) and 1 mM EDTA. T2-T4 buffer contained 4 g/lNaCl, 5 g/l K2SO4, 1.5 g/l KH2PO4, 3.52 g/l Na2HPO4,1 mM MgSO4, 0.1 mM CaCl2 and 1 % (w/v) gelatine.SDS-sample buffer (3�) for denaturing PAGE contained150 mM Tris-HCl (pH 6.8), 6 mM EDTA, 3 % (w/v) SDS,

3 % (v/v) 2-mercaptoethanol, 24 % (v/v) glycerol and0.075 % (w/v) bromphenol blue.

Chemicals and radiochemicals

Ethanolamine was from Sigma (Deisenhofen,Germany). PEN-Bristol ampicillin-sodium was pur-chased from GruÈ nenthal (Stolberg, Germany).[g-32P]dATP (speci®c activity >5000 Ci/mmol) was pur-chased from Amersham (Braunschweig, Germany).Acrylamide/bisacrylamide premix (29:1, w/w) for dena-turing PAGE and polyethyleneglycol 6000 (PEG-6000)were from Serva (Heidelberg, Germany). Dextran T500(Dex-500) was purchased from Pharmacia (Freiburg,Germany). Acrylamide/bisacrylamide premix (37.5:1,w/w) for SDS-PAGE was from BioRad (MuÈ nchen,Germany). All other chemicals were purchased fromMerck (Darmstadt, Germany). The chromatographicmaterial and columns were purchased from Pharmacia(Freiburg, Germany).

Proteins and enzymes

endo VII of phage T4 was puri®ed in our laboratoryfrom overexpressing bacteria as described (Golz et al.,1995). Strand transfer helper protein UvsY of phage T4was puri®ed in our laboratory from overexpressing bac-teria as described (Birkenkamp-Demtroeder et al., 1997).Proteins gp16, gp170 and gp20 were puri®ed from over-expressing bacteria as described here. The phage T7polymerase sequencing kit and puri®ed gp32 were pur-chased from Pharmacia (Freiburg, Germany).

Oligonucleotides and DNA substrates

Synthetic oligonucleotides were purchased from Phar-macia (Freiburg, Germany) and puri®ed by preparativedenaturing PAGE on 10 % gels. Hybrid substrates wereprepared by the annealing procedure as described(Birkenkamp-Demtroeder et al., 1997). PCR primers forcloning genes 16 and 20 were: gene 16, 50-TTAAGGCGC-CATATGTGATTAATCGGTTGT-30 and 50-TAACCC-GGGCATATGGAAGGTCTTGATATAAAC-30; gene 20,50-AATTCCCCTCTAGAAATAATTTTGTTTAACTTTA-AGAAGGAGATATACATATGAAATTTAATGTATTA-AG-30 and 50-TTAGCAGCCGGATCCCTATTAAAAAT-CCTCTTG-30.

PCR reactions and cloning

PCR reactions were performed in a total volume of100 ml reaction buffer indicated by the manufacturer, 100pmol of primer DNA, 100 ng genomic template DNA,2 mM MgCl2, 160 (M each of dNTPs (Pharmacia, Frei-burg, Germany) and 1 unit of Taq polymerase (Appli-gene, Heidelberg, Germany). Initial denaturation was at94 �C for ®ve minutes, followed by 26 cycles of 94 �C forone minute, 55 �C for one minute and 72 �C for one min-ute and a ®nal elongation step for ®ve minutes at 72 �C.The PCR products were puri®ed by spin column cen-trifugation (Qiagen, Hilden, Germany), digested withtherestriction enzymes NdeI (for cloning of gene16) orBamHI/XbaI (for cloning of gene 20) and extracted withphenol/chloroform before ligation into the NdeI-digestedor BamHI/XbaI-digested and dephosphorylated vectorpET11-a, respectively. Ligation products were ®rst trans-formed into E. coli DH5a and the correct sequence ofgenes 16 and gene 20 was veri®ed by dideoxy sequen-


cing (Sanger et al., 1977) using the T7 polymerasesequencing kit. Plasmid DNA was then transformed intothe E. coli expression strain BL21(DE3) by heat shock(Sambrook et al., 1989).

Purification of proteins gp16, gp170 and gp20

All manipulations were carried out at 4 �C or on ice ifnot stated otherwise. Crude extracts were prepared from5 g of frozen cells. Cells were thawed on ice, resus-pended in four volumes (v/w) of buffer A for gp16 orbuffer BATP

30 (subscript numbers indicate concentrationsof KCl in mM) for gp170 and sonicated for 30 minuteswith a Branson soni®er equipped with a 0.5 cm tip andsetting of 5 until the A578 was reduced to 0.3. Puri®cationwas monitored by optical equipment and purity of pro-tein fractions was determined by SDS-PAGE followed bysilver staining (Heukeshoven & Dernick, 1988).

Purification of gp16

The sonicated extract was centrifuged in a Beckmanultracentrifuge using a SW50.2 Ti-rotor at 100,000 g for30 minutes to remove cell debris. The supernatant wastreated with a PEG/dextran two-phase separation understandard conditions as described (Golz et al., 1995). Theseparated, protein-enriched PEG phase was collectedand diluted with an equal amount of buffer B200 and dia-lysed overnight against the same buffer.

Portions (25 ml) were loaded onto a 5 ml HiTrapQcolumn equilibrated with buffer B200. The column waswashed with ten column volumes of the same buffer fol-lowed by ®ve column volumes of a linear gradient ran-ging from B200 to B1000. Fractions of 1 ml were collected.gp16 eluted at a salt concentration of 340-500 mM KCl.The fractions containing the highest gp16 concentrationwere loaded on a Superose 12 column without dialysis.Gel ®ltration was performed with 50 ml of buffer C50 ata ¯ow-rate of 0.25 ml/minute and a fraction size of0.5 ml. gp16 elutes in the ®rst sharp protein peak. Thegp16-containing fractions were pooled and loaded onto a1 ml HiTrap Heparin column without further dialysis.The chromatography was performed with buffer C50 at a¯ow-rate of 0.5 ml/minute. The ¯ow-through, contain-ing gp16, was pooled and dialysed against buffer B100

overnight. The dialysed extract was loaded onto aMonoQ HR5/5 column with a ¯ow-rate of 0.5 ml/min-ute. The column was washed with 25 column volumesof the same buffer followed by ®ve column volumes of alinear gradient from B100 to B1,000. Fractions of 250 mlwere collected. gp16 eluted in a sharp peak at 350 mMKCl. The fractions were dialysed against buffer B20 andstored at ÿ80 �C. This was the ®nal protein solution, con-taining gp16 more than 99.99 % pure (Figure 1, lane 2).Nine C-terminal amino acid residues are deleted fromgp16 by the action of an E. coli protease active in phageinfected as well as overexpressing cells (Lin et al., 1997).The major fraction of the puri®ed protein consists of thetruncated variant visible in the lower band of the twobands on the gel (Figure 1, lane 2). The preparation wasfree from detectable exo- or endonucleolytic activities,and up to 250 ng of the puri®ed protein did not degrade32P end-labelled double-stranded or single-strandedDNA as tested under endo VII assay conditions.

Purification of gp17 0

gp170 was puri®ed following a published procedure(Rao & Black, 1988) with the following modi®cations.The sonicated extract obtained from overexpressing bac-teria was centrifuged in a Beckman ultracentrifuge usinga SW50.2 Ti-rotor at 100,000 g for 30 minutes to removecell debris and dialysed against buffer BATP

30 . For all chro-matographic steps, a ¯ow-rate of 0.5 ml/minute wasused. Portions (10 ml) were loaded onto an 5 mlHiTrapQ column equilibrated with the same buffer. Thecolumn was washed with ten column volumes of thesame buffer followed by ®ve column volumes of a lineargradient ranging from BATP

30 to BATP1,000 mM KCl. Fractions

of 1 ml were collected; gp170 eluted between BATP150 and

BATP250 . The gp170-containing fractions were pooled and

dialysed against buffer BATP200 . The dialysed extract was

loaded onto a 1 ml HiTrap Blue column. After washingthe column with 30 ml of equilibration buffer, the con-taminating proteins were eluted with a linear gradientreaching from BATP

200 to BATP1,800. The column was then

washed with 30 ml of buffer BATP1800 followed by a washing

step of 10 ml of buffer BATP50 . gp170 was eluted in a ®nal

step with buffer BATP50 containing 50 mM ATP instead of

1 mM ATP. This was the ®nal protein solution withgp17 more than 99.99 % pure (Figure 1, lane 4). Afterpuri®cation it was noted that the truncated version,gp170 with a molecular mass of 59 kDa, had been puri-®ed instead of the full-size protein gp17 with a mol-ecular mass of 68 kDa. The smaller protein appearsfrequently in extracts from expression plasmids togetherwith another truncation, gp1700. Both proteins are poss-ibly made from internal in-frame ribosome binding sitesof gene 17 (Franklin & Mosig, 1996). Protein gp170 wasused throughout this study.

Purification of gp20

The sonicated extract was centrifuged in a Beckmanultracentrifuge using a SW50.2 Ti-rotor at 100,000 g for30 minutes to generate a pellet containing gp20. The pel-let was carefully washed by ®ve repeated cycles of resus-pending in 5 ml of buffer AU containing 2 M ureafollowed by low-speed centrifugation to collect theremaining pellet. The washed pellet was then resus-pended in buffer AU (20 ml/g pellet) containing 4 Murea. The suspension was stirred for one hour at roomtemperature and then centrifuged for 30 minutes at14,000 rpm in a Sorvall SM24 rotor. The supernatant wasdialysed against buffer AU

300 containing 4 M urea. Por-tions 5 (ml) were loaded onto a MonoQ HR5/5 columnwith a ¯ow-rate of 0.5 ml/minute. The column waswashed with 10 ml of equilibration buffer. For renatura-tion, a descending linear gradient of 80 ml containingurea from 4 M to 0 M in buffer AU was applied. A ®nalwashing step of 30 ml of buffer A300 without urea fol-lowed. The protein was ®nally eluted with a 5 ml linearsalt gradient reaching from A300 to A1000 and gp20 elutesin a single peak between A300 and A360. This solutioncontained gp20 more than 99.99 % pure (Figure 1, lane5).

endo VII-affinity chromatography

endoVII-Sepharose for af®nity chromatography wasprepared by coupling 1 mg of highly puri®ed endo VIIto 1 ml of HiTrap NHS-activated Sepharose as described(Birkenkamp-Demtroeder et al., 1997). All chromatog-raphy steps were carried out at 4 �C. endoVII af®nity col-umns were equilibrated with 5 ml of buffer BATP

30 at


pH 8.0. Samples of proteins gp16, gp170 or gp20 weredialysed against buffer BATP

30 at ®nal concentrations of40 ng/ml each. Proteins in a total volume of 500 ml wereloaded onto the endo VII-af®nity column with a ¯owrate of 0.2 ml/minute. Columns were kept for 20 min-utes without ¯ow and then washed with 5 ml of bufferBATP

30 . Adsorbed proteins were eluted with a 5 ml lineargradient from BATP

30 to BATP1000. Elution of proteins was mon-

itored by measuring simultaneously the conductivityand the absorbance at 280 nm.

Gel filtration

Gel ®ltration was performed with Superose 6 or 12columns depending on the molecular mass of the proteincomplexes to be analysed. For gel ®ltration experiments,25 mg of each protein was mixed in a total volume of100 ml of buffer BATP

100 and preincubated for 15 minutes at16 �C. The protein complexes were chromatographed inbuffer BATP

100 at a ¯ow-rate of 0.25 ml/minute at 4 �C.Fractions of 0.5 ml were collected and the absorbance at280 nm was measured.

Enzyme-linked immunosorbent assay (ELISA)

Sandwich-ELISAs were done according to the labora-tory manual with modi®cations (Harlow & Lane, 1988).Wells of 96-well microtiter plates were coated with 50 mlof 0.5 � PBS containing ®nal concentrations of 10 mg/mlof gp20 or 5 mg/ml of endo VII. Plates were stored over-night in a humid atmosphere at 4 �C. After extensivewashings (5�) with 200 ml of 0.5 � PBS per well, 200 mlof blocking buffer was added and incubated for onehour at room temperature. After another series of exten-sive washings, 50 ml of endo VII in 0.5 � PBS was addedand incubated for one hour at room temperature. Afterwashing with 50 ml of an antibody solution containingpolyclonal rabbit anti-endoVII antibodies, diluted 1:5000in 0.5 � PBS containing 0.5 % BSA, was added and incu-bated for 45 minutes at 37 �C in a humid atmosphere.The amount of endo VII bound to the respective immobi-lised proteins corresponds to the amount of bound anti-endo VII antibody and was determined indirectly with asecond antibody conjugate. After a washing step, 50 mlof an antibody dilution of goat anti-rabbit IgG (H � L)-AP conjugate coupled to alkaline phosphatase wasadded and incubated for 45 minutes at 37 �C. Afterextensive washings, 50 ml of ALP dye was added andincubated for ®ve minutes at room temperature. Reac-tions were stopped with 0.1 M NaOH and absorbancewas measured in a microtiter plate reader at 405 nm.

Electrophoretic mobility shift assay (EMSA)

Reactions were carried out in 10 ml of TEDB-buffercontaining 2 fmol of radioactively labelled DNA andvarying amounts of protein. Protein was added after themixtures were pre-incubated for ®ve minutes on ice toremove traces of Mg2�. Reactions were incubated for 15minutes at 16 �C and terminated by addition of 5 ml ofLB-buffer (5�). Samples were loaded onto 0.75 mmthick, native 12 % polyacrylamide gels in a BioRad Mini-protean II chamber and electrophoresed in NE buffer forthree hours at 4 �C, 7 V/cm, according to Parsons et al.(1990). Reaction products were visualised and quanti®edby phosphorimaging.

Gradient centrifugations

Linear glycerol gradients were prepared using a gradi-ent mixer from Pharmacia (Freiburg, Germany). Thesamples were centrifuged for 17 hours at 15 �C in aSW41 Ti rotor at 28,000 rpm. Fractions of 250 ml werecollected from top to bottom of the gradient. Sampleswere with trichloroacetic precipitated acid, analysed onSDS/polyacrylamide gels, blotted on Hybond-Csuper

membrane and stained with Ponceau S. The followingproteins used as sedimentation markers were purchasedfrom Sigma (Deisenhofen, Germany): bovine albuminserum (66 kDa), alcohol dehydrogenase from yeast(150 kDa), b-amylase from sweet potato (200 kDa), apo-ferritin from horse spleen (443 kDa), bovine thyroglobin(669 kDa) and blue dextran (2000 kDa).

Enzyme reactions

If not stated otherwise, reactions contained 2 fmol ofradioactively labelled DNA, endo VII reaction mix andproteins gp16, gp170, gp20 or gp49 in amounts as indi-cated. Reaction mixtures were incubated at 37 �C for 15minutes. Samples were deproteinized by addition of10 ml of proteinase K (20 mg/ml) and incubated for 30minutes at 37 �C. After precipitation in ethanol, sampleswere resuspended in M&G stopmix and loaded ontodenaturing 15 % polyacrylamide gels. Reaction productswere visualised by autoradiography and quanti®ed byphosphorimaging. The enzymatic activities of gp16 andgp170 were tested exactly as described (Rao & Black,1988).

Other procedures

Protein concentrations were determined according toBradford (1976) using a kit from BioRad (MuÈ nchen,Germany) with BSA as standard. SDS-PAGE was per-formed using the buffer system of Laemmli et al. (1970)in a BioRad Miniprotean II device and silver stainingwas done according to Heukeshoven & Dernick (1988).Quanti®cation of radioactive samples on gels was doneusing a phosphorimager BAS 1000 from Fuji. All chro-matographic procedures were performed using FPLCequipment from Pharmacia (Freiburg, Germany).

Acknowledgements

This work was supported through funds from theDeutsche Forschungsgemeinschaft (NormalverfahrenKe188/8) and ``Fond der Chemischen Industrie'' givento B. K. We are indebted to Gisela Mosig, who donatedthe plasmid pJK17. We gratefully acknowledge the tech-nical assistance of Vera Illner-BraÈutigam and the cricticalreading of the manuscript by Libby Guethlein from ourInstitute.

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Edited by J. Karn

(Received 13 July 1998; received in revised form 13 November 1998; accepted 13 November 1998)


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