Compression of the DNA Substrate by a Viral Packaging Motor Is Supported by Removal of Intercalating Dye During Translocation

Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation

Aparna Banerjee Dixita, Krishanu Raya,b, and Lindsay W. Blacka,1

aDepartment of Biochemistry and Molecular Biology and bCenter for Fluorescence Spectroscopy, University of Maryland School of Medicine, Baltimore, MD 21201

Edited* by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved October 25, 2012 (received for review August 20, 2012)

Viral genome packaging into capsids is powered by high-force- widely used because of its over 1,000-fold higher affinity to dsDNA generating motor proteins. In the presence of all packaging compo- than EthBr. It binds virtually irreversibly to the DNA, and the fl nents, ATP-powered translocation in vitro expels all detectable uorescence quantum yield of the bound dye is more than 1,000- fold higher than when free in solution. As a result, the background tightly bound YOYO-1 dye from packaged short dsDNA substrates fl and removes all aminoacridine dye from packaged genomic DNA in uorescence from free dye is extremely low, which makes these dyes excellent probes for high-sensitivity quantification of DNA vivo. In contrast, in the absence of packaging, the purified T4 – ∼ and for imaging of individual DNA molecules (13). At low dye packaging ATPase alone can only remove up to 1/3 of DNA-bound base pair ratios, the binding mode appears to consist primarily of intercalating YOYO-1 dye molecules in the presence of ATP or ATP- bisintercalation. Each monomer unit intercalates between adja- γ-S. In sufficient concentration, intercalating dyes arrest packaging, cent bases, with the benzazolium ring system sandwiched between but rare terminase mutations confer resistance. These distant muta- the pyrimidines and the quinolinium ring between the purine rings, tions are highly interdependent in acquiring function and resistance causing the helix to unwind as studied by NMR. Important and likely mark motor contact points with the translocating DNA. In structural parameters such as the binding site size, the elongation, stalled Y-DNAs, FRET has shown a decrease in distance from the as well as the untwisting angle per bound YOYO-1 molecule have phage T4 terminase C terminus to portal consistent with a linear been reported (14). In a recent study, YOYO-1 dye was used to BIOCHEMISTRY motor, and in the Y-stem DNA compression between closely posi- monitor the helicase activity of the Bacteriodes fragilis AddAB enzyme through its displacement from the unwound dsDNA (15). tioned dye pairs. Taken together with prior FRET studies of confor- fi ∼ – mational changes in stalled Y-DNAs, removal of intercalating We are able to measure DNA packaging at high ef ciency ( 20 100%) in vitro with short DNAs ranging from 70 bp to 5 kb, as well compounds by the packaging motor demonstrates conformational as with full-length ∼170 kb genome size DNAs that can produce change in DNA during normal translocation at low packaging re- “ ” infectious virions. We quantify packaging in vitro by independent sistance and supports a proposed linear DNA crunching or tor- nuclease, Typhoon imager, and fluorescence correlation spectros- sional compression motor mechanism involving a transient grip- copy (FCS) assays. We report here that the purified bacteriophage and-release structural change in B form DNA. T4 packaging ATPase reduces ID binding to DNA in the absence of ATP hydrolysis and proheads. Unexpectedly, we find that DNA DNA structure | terminase inhibitors translocation expels all detectable YOYO-1 from the packaged DNA substrate. We observe similar inhibitory effects in vitro of ucleic acid translocation into an empty procapsid is a con- several IDs on packaging of DNAs of different low molecular served capsid assembly mechanism found among diverse weights as well as full-length genomic DNAs in vivo. Thus, we N expect that ID inhibition should reflect effects on local DNA DNA and RNA viruses (1). High-resolution structures of all of the fi conserved motor components found among tailed dsDNA bac- structure. Moreover, we nd that terminase mutations confer teriophages have been determined (2–5). The small bacteriophage comparable resistance to the different IDs 9AA and EthBr in vivo and 9AA, EthBr, and YOYO-1 in vitro. By mutagenic treatment T4 terminase protein gp16 is required for cutting and packaging and site-directed mutagenesis (SDM) we show complex in- the replicative DNA concatemer in vivo but is nonessential and terdependence of three rare mutational sites within the terminase inhibitory for linear DNA packaging in vitro (6). Thus, T4 DNA to confer resistance to IDs and to maintain terminase function in translocation in vitro can be driven by a two-component motor the absence of IDs. These results are consistent with the structure consisting of a prohead portal ring channel dodecamer situated at and proposed mechanism of the DNA packaging motor. a single packaging vertex that is docked during packaging with gp17 terminase-ATPase. A linear packaging motor mechanism Results proposed that a terminase to portal DNA grip-and-release driven by a motor protein conformational change drives DNA into the T4 Terminase Acridine-Resistant Mutants Resist ID Inhibition of Growth prohead by a DNA compression motor stroke (7–10). and of DNA Packaging. Previously IDs like 9AA and EthBr have It has long been known that acridine dyes inhibit bacteriophage been shown to inhibit bacteriophage development at concen- development at concentrations below those that inhibit growth of trations below those that inhibit the bacterial host. Two 9AA re- the bacterial host. Phage mutations that confer resistance to such sistant mutants, ac and acq, the latter a double mutation conferring dyes arise through different mechanisms. Among these are mu- additional resistance to the intercalating compound quinacrine, tations in the phage T4 terminase gene 17, called ac and q, that were isolated after intensive selection and screening and were confer acridine and quinacrine resistance (11). All 9-amino- found to be located in the terminase gene 17 by genetic mapping acridine (9AA) treatments have been shown by electron micros- copy to arrest DNA packaging in vivo; however, whether the site of action of 9AA is the DNA, the active site of the packaging enzyme, Author contributions: A.B.D., K.R., and L.W.B. designed research; A.B.D., K.R., and L.W.B. or the enzyme DNA complex—in fact, acridines are known to performed research; A.B.D., K.R., and L.W.B. analyzed data; and A.B.D. and L.W.B. wrote inactivate protease active sites or virion proteins associated with the paper. DNA (reviewed by Black et al., in ref. 12)—is unknown. The authors declare no conflict of interest. Insertion of intercalating dyes (IDs) between dsDNA base pairs *This Direct Submission article had a prearranged editor. has been intensively studied and is known to cause stretching and 1To whom correspondence should be addressed. E-mail: [email protected]. unwinding of DNA. Classic IDs are plane aromatic molecules such This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. as 9AA and ethidium bromide (EthBr). Dimeric ID YOYO-1 is 1073/pnas.1214318109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1214318109 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 (11). The locations of the two terminase mutations in the 17 gene YOYO-1–bound 70 bp, 280 bp, and 5 kb DNAs with and without were determined to be ac-A96D and q-F249V. The mutations en- terminase could be fitted by a single-species diffusion model, con- hanced T4 phage growth by a factor of 107 (Fig. 1A). When ex- sistent with a single fluorescent species. The diffusion coefficients of amined for growth inhibition by different IDs, 9AA and EthBr, acq the YOYO-1–bound 70 bp, 280 bp, and 5 kb DNAs without ter- showed greater resistance in comparison with the ac mutant (Fig. minase were around 70 μm2/s, 20 μm2/s, and 2 μm2/s and with ter- 1B). When DNA packaging was determined by growth in the minase were 85 μm2/s, 30 μm2/s, and 3 μm2/s, respectively (Table 1). presence of 9AA (3 μg/mL), packaging was inhibited in wild-type A fluorescence intensity decrease of ∼5 counts/ms was observed in (wt) T4 phage, where consistently we saw only partial (∼10 kb) the presence of terminase for YOYO-1–bound 70 bp DNA (Fig. packaging of the full ∼170 kb genomic DNA. The ac mutant pro- 3B), and similar fluorescence intensity decrease was also observed duced mostly full genomic T4 DNA along with a lesser amount of for YOYO-1–bound 280 bp and 5 kb DNAs (Fig. S1). A decrease in the 10 kb DNA, whereas the acq mutant was found to be resistant to fluorescence intensity was also seen with YOYO-1–bound 5 kb the inhibition of DNA packaging and no 10 kb DNA was seen (Fig. DNA in the presence of terminase by agarose gel Typhoon image 1B). Interestingly, glycerol gradient purified partially or completely analysis of fluorescence (Fig. 3C). The dye removal by the terminase filled heads from these infections in the presence of 9AA did not alone is in the range of 18–35% as calculated from decrease in the show any 9AA fluorescence by Typhoon imager analysis of the photon counts for our FCS experiments (Table S1). This analysis intact and partially filled heads on an agarose gel (Fig. 1C). shows that there is an increase in the diffusion coefficients and decrease in fluorescence intensities of the YOYO-1–bound DNAs IDs Inhibit in Vitro DNA Packaging and Acridine-Resistant Mutant in the presence of terminase, clearly suggesting that terminase can Terminases Are More Resistant to in Vitro DNA Packaging Inhibition release some amount of ID YOYO-1 from DNA. Fig. 3C shows Compared with the wt Terminase. In this study we have used partial removal of YOYO-1 by terminase alone, which is enhanced YOYO-1 dye, which has higher DNA affinity and higher fluores- inthepresenceofATPorATP-γ-S. Analysis of gp17/DNA–dye cence quantum yield than EthBr homodimer, 9AA, DAPI, and binding in the presence or absence of ATP (Fig. S2) shows that Hoechst dyes and only fluoresces after binding to DNA. All three when gp17 binding to the DNA reaches saturation, only partial dye IDs (9AA, EthBr, and YOYO-1) were inhibitory for in vitro DNA release is observed, although dye release is enhanced by ATP or packaging at a concentration of ∼1 μM (Fig. 2A). At 2.5 μMcon- ATP-γ-S even in the absence of translocation into the proheads. centration of 9AA or YOYO-1 wt T4 packaging was 100% inhibited, whereas the acq mutant was only about 50% inhibited DNA Translocation Removes IDs from the DNA–Dye Substrate Complex. (Fig. 2D). Nuclease assays in the presence (+) and absence (–)of When 5 kb DNA equilibrated with different concentrations of YOYO-1 with equal amounts of purified wt, ac,andacq terminases YOYO-1 (250 nM, 500 nM, and 1 μM) was packaged in vitro, as (Fig. 2B) showed that the acq terminase is more resistant to the expected, less packaging of DNA was observed with increasing inhibition than the wt and the ac mutant (Fig. 2 C and D). YOYO-1 concentrations of dye, but unexpectedly, no fluorescence was ob- and 9AA dyes inhibited packaging in the presence of wt and acq served in the packaged DNA. This was determined by Typhoon mutant terminases in a similar manner as shown in Fig. 2D. images of intact proheads in an agarose gel before and after EthBr staining (Fig. 4A), suggesting that the DNA packaged into the Terminase Releases IDs from DNA in the Absence of Translocation. prohead does not have any bound YOYO-1 dye. Some back- Upon electrophoresis, YOYO-1–bound DNAs ran higher on an ground fluorescence was seen by Typhoon image analysis when agarose gel compared with the unbound DNA, suggesting a change proheads alone were incubated with 1 μM YOYO-1, even though in the conformation and/or molecular weight of the DNA–dye there is no DNA in the proheads as seen in the nuclease assay gels complex (Fig. 3A). When the YOYO-1–bound DNAs (70 bp, 280 (Fig. 4A). To verify this result, packaging of smaller DNAs (70 bp bp, and 5 kb) were incubated with wt terminase at 37 °C for 30 min, and 280 bp) was carried out and checked by nuclease assays as well an increase in DNA mobility was observed by agarose gel elec- as by FCS measurements (Fig. 4 B and C). The FCS autocorre- trophoresis analyzed on Typhoon imager and EthBr staining (Fig. lation curves of the YOYO-1–bound 70 bp and 280 bp DNA 3). Similar results were observed in FCS experiments, when the substrates in a negative reaction control lacking proheads could three different lengths of YOYO-1–bound DNAs (70 bp, 280 bp, be fitted by a single-species diffusion model, consistent with sin- and 5 kb) were incubated with terminase. The autocorrelation of the gle fluorescent species with diffusion coefficients of about 85 μm2/s

Intercalating dye wt ac acq

No dye 1 1 1 9AA 9AA 1 kb DNA ladder acq ac T4 Phage DNA 9AA T4 1 kb DNA ladder 9AA (5 nM) ~ 1.2 x 10-7 11 Lambda DNA ladder

EthBr (2.5 nM) ~ 0.5 x 10-7 11

200 kb 150 kb C i ii 50 kb 10 kb

9AA heads 9AA 5kb 9AA heads 9AA ac T4 9AA heads T4 phage DNA acq Fig. 1. T4 terminase acridine-resistant mutants are 1 kb DNA ladder more resistant to inhibition of growth as well as 3kb packaging of DNA by IDs. (A)Efficiency of plating of wt and mutant T4 phages with and without IDs (9AA and EthBr). Pulse field gel electrophoresis (PFGE) (B)and 10 kb agarose (C) gels showing different-length DNAs packaged in the presence of 9AA in T4 wt and acridine- resistant ac and acq mutants. Typhoon image of the agarose gel before (C, ii) and after EthBr staining (C, i) Free 9AA showing no DNA-bound 9AA in the glycerol gradient- purified proheads.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1214318109 Dixit et al. Downloaded by guest on October 1, 2021 AB 9AA Input DNA 1kb DNA ladder No dye YOYO1 No gp17 EhtBr Wt type gp17 type Wt ac gp17 acq gp17 95 kDa

72 kDa 5kb

55 kDa

C D ac gp17 acq gp17 - + - gp17 type Wt + - +

Fig. 2. IDs inhibit in vitro DNA packaging. (A) Nu- clease assays showing inhibition of packaging in the μ 5kb presence of YOYO-1, 9AA, and EthBr dyes (1 M). (B) SDS/PAGE gel image showing puriﬁed ac, acq,and wt terminases. (C) The acq terminase mutant more resistant to packaging inhibition by 9AA (500 nM) compared with ac and wt. (D) YOYO1 and 9AA dyes inhibit DNA packaging in a similar manner, and acq is more resistant to inhibition than the wt terminase.

μ 2 – and 30 m /s, respectively (Fig. 3B and Table 1). The FCS cor- dsDNA, which in some way releases more dye from the dye DNA BIOCHEMISTRY relations obtained in the full packaging reactions were apparently complex compared with the wt terminase during packaging. identical and showed no prohead-like diffusion, as reported earlier 2 with a slow species of diffusion coefficient of ∼1–3 μm /s (10), and ac, q, and N Sites Coordinate with Each Other and May Be Near fi could be tted to the same fast diffusing species as shown in our Terminase DNA Binding Sites. We determined that ac and q each previous studies of covalently dye-labeled dsDNAs of this size confer 9AA resistance as single mutations, but more effectively range (Fig. 4C). The analysis showed further that even though ef- fi when combined, and by mutagenesis that such mutational sites in cient packaging of the DNAs was seen by nuclease assay gels (Fig. the terminase are rare. In fact, we were unable to isolate a single 4B), no prohead-like diffusion of the packaged DNA was observed additional 9AA resistance mutation comparable to the ac and q in the FCS assays (Fig. 4C), thus confirming the Typhoon results mutations (ac and q are transversion mutations) at other sites in showing that the terminase in the presence of the complete packaging mix completely removes the YOYO-1 intercalated dye from gene 17 following heavy hydroxylamine mutagenesis of plasmid the complex and the DNA without dye is packaged into the pro- gene 17 followed by strong selection for recombinant mutant heads. Quenching of YOYO-1 in the DNA packaged proheads is genomes. Also by screening for spontaneous mutations, we found inconsistent with the observation that YOYO-1 can slowly diffuse only identical substitutions at the same sites. However, mutagenesis into the proheads and bind to the packaged DNA judged by the identified a third interacting site, N267, that modified ID resistance Typhoon gel analysis (Fig. S3). The wt and acq terminases alone at the ac and q sites. Introduction of single and multiple amber were comparable in releasing dye from the YOYO-1 DNA complex codons at the ac, q,andN sites by SDM allowed us to determine the (Fig. 4 D, i), whereas in the full packaging mix acq terminase re- viability and 9AA resistance of 13 different single and double leased significantly more dye (Fig. 4 D, ii). The mutations in the ac amino acid replacements at these three sites. This analysis shows and acq terminases may result in tighter binding to the translocating that the three sites are highly cooperative and interdependent both

AB C -S γ - γ -S S -S γ - γ - ATP 70bp DNA+dye DNA DNA+dye+gp17

70bp+Terminase

500 bp 250 bp 70 bp DNA+dye+3 gp17/100bp-ATPDNA+dye+3 DNA DNA+dye gp17/1kb+ATPDNA+dye+2 gp17/1kb+ATPDNA+dye+8 gp17l/100bp+ATP DNA+dye+3 gp17/100bp+ATPDNA+dye+12 DNA+dye+12 gp17/100bp+DNA+dye+12 ATP DNA+dye+8 gp17/1kb-ATPDNA+dye+8 DNA DNA+dye gp17/1kb+DNA+dye+2 ATP- DNA+dye+8 gp17/1kb+DNA+dye+8 gp17/100bp+DNA+dye+3 ATP- DNA+dye+2 gp17/1kb-ATPDNA+dye+2 gp17/100bp-ATPDNA+dye+12 i Fig. 3. Terminase releases IDs from DNA. (A)Gel mobility shift assays showing the shift in YOYO-1– bound (500 nM) 70 bp, 280 bp, and 5 kb DNAs in the 750 bp presence of terminase. (B) Decrease in ﬂuorescence Typhoon image 250 bp intensity of the 70 bp DNA–YOYO-1 complex in the 280 bp ii presence of terminase observed in the FCS measurements. (C) Mobility shift and reduction in ﬂuorescence intensity of YOYO-1–bound 5 kb DNA by terminase in 10 kb EthBr stained the presence or absence of ATP or γ-S-ATP in the re- image 5 kb action mixture (C, i—Typhoon image) but no change in 5 kb DNA concentration (C, ii—EthBr stained gel).

Dixit et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 Table 1. FCS diffusion coefficients of the DNA–dye complexes Discussion fi Diffusion coefficients The principal nding of our study is that DNA packaging expels the Sample with 500 nM YOYO-1 (D) (μm2/s) IDs 9AA, EthBr, and YOYO-1 from DNA in vivo and in vitro. DNA saturating levels of T4 terminase remove ∼18–35% bound 70 bp DNA 70 ± 6 YOYO-1 from the equilibrated dye–DNA complex in the absence 70 bp DNA + terminase alone or 85 ± 8 of proheads and ATP-powered translocation. The terminase is with full packaging mix qualitatively different with all packaging components present 280 bp DNA 20 ± 1 during translocation in eliminating all detectable YOYO-1 bound 280 bp DNA + terminase alone or 30 ± 2 to even very short DNAs (70 bp or 280 bp) that are packaged. with full packaging mix Resistance to packaging of short DNAs from DNA pressure 5 kb DNA 2 ± 0.1 within the prohead is expected to be low (17, 18). It is thus unlikely that it is the packaged conformation of the DNA that quantita- 5 kb DNA + terminase alone 3 ± 0.2 tively removes the dye but rather the translocation itself; indeed, Values of the DNA–dye complexes alone, with terminase, or following the packaged DNA in the intact prohead can be readily stained packaging in the full packaging mix are given as average ± SE as previously with EthBr and also YOYO-1 (Figs. 1 and 4 and Fig. S3). Removal determined (refs. 8 and 10). of the IDs during translocation is at first sight surprising, as the T4 motor can efficiently package DNA labeled with dye covalently attached to the DNA ends or to the bases (19). The Φ29 packaging in allowing terminase function itself in the absence of IDs and also ATPase is also packaging tolerant of heavily modified duplex in producing ID resistance; for example, we were able to introduce DNA (20). And although ID removal is expected and in fact shown an amber codon at N only following a mutation at q that changed F for a helicase that separates the DNA strands, this is not expected to L as shown by the arrow in Fig. 5A. Although this substitution for a packaging translocase that moves intact B form DNA from allowed an Nambercodon to be introduced, it could be grown only outside to inside the prohead. However, this surprising observa- with Y or F substitution for W at this site, and these changes ren- tion is consistent with previous observations including the effect of dered the terminase ID sensitive, whereas the single q site L was nicking short DNA substrates, which was demonstrated to disen- resistant. Additionally, the combination L249, L96 was dye resistant, gage the DNA from the motor (7). This finding and others led to whereas the single L96 substitution was sensitive; similarly, 96Y a proposal that a linear DNA grip-and-release motor mechanism conferred viability on the otherwise lethal 249Y substitution. Some transiently compresses B form DNA during translocation. In fact, of the interdependencies of the ac, q,andN sites for ID resistance previous FRET studies of stalled Y-DNAs directly supported and terminase function are summarized in Fig. 5A. Although com- torsional compression of the Y-stem duplex by 20–25% (8, 10). plex, these results show that these three sites coordinately participate These studies supported the proposed compression motor mech- in the same terminase function. The location of the three sites in the anism of DNA translocation and also showed that conformational gp17 crystal structure (4) is shown in Fig. 5A. changes in both the motor proteins and the DNA substrate itself Various temperature-sensitive (ts) mutations affecting DNA are associated with the power stroke of the packaging motor. Our translocation were previously located at the C-terminal end of present studies support such a conformational change in the DNA gp17 (reviewed in ref. 16 or newly isolated by us, in Materials and that is apparently found not only in stalled Y-DNA but also ac- Methods). Two ts mutants showed changes at residue Y562. One companies active translocation of linear DNA into the prohead. is a deletion mutant, and in the other, Y is substituted by A. The Compression of the B form duplex would be predicted to expel ts mutation at residue Y562 of gp17 (Y deletion) is suppressed by intercalating compounds from between the base pairs, as it is es- E573K mutation in the same gene. One ts mutation, S546G, was tablished that insertion of IDs between bases unwinds, stretches, suppressed by a distant D584N mutation. Another ts mutation, and stiffens the DNA without affecting the bending rigidity (14). D473N, was suppressed by portal cold-sensitive (cs) mutation Indeed, it has been shown that YOYO-1 binding is very stable and csM308I located in the clip interaction region (Fig. 5B). All these is only affected by stretching forces exceeding 10 pN (14); thus, the mutations in the C-domain of the gp17 gene affecting the DNA complete removal of IDs from DNA without changing the integ- translocation suggest its active participation in DNA trans- rity of the DNA appears most probably caused by an opposing location in conjunction with the portal clip region (3, 10). compression of the duplex by the packaging motor that can

A B C D +1µM dye +1µM wt dye gp17+1µM i DNA DNA+dye acq dye gp17+1µM No gp17 Input DNA Input Packaged DNA Prohead Pmix+1µM dye Pmix+1µM Pmix+250nM dye Pmix+250nM Pmix+500nM dye Pmix+500nM i DNA Input Typhoon image Prohead Fig. 4. DNA is packaged after removal of ID from 5 kb the DNA–dye complex. (A) Packaging of 5 kb DNA in DNA-dye 70 bp the presence of different concentrations of YOYO-1. ii EthBr stained (A, i) Typhoon image, (A, ii) same gel stained with image EthBr, and (A, iii) nuclease assay gel. (B) Nuclease assay gels showing packaging of the 70 bp and 280 ii bp DNAs in the presence of YOYO-1 (500 nM). (C)FCS Input Input DNA No gp17 Packaged DNA data showing ﬁtting to a single fast diffusing component (DNA–dye complex) and no prohead-like iii gp17 gp17+1µM dye gp17+1µM diffusion of the YOYO-1–bound 70 bp and 280 bp DNA in the full packaging mixture (Pmix). (D, i)Ty- Pmix+wt DNA+dye Pmix+acq gp17 Pmix+wt Pmix+acq dye gp17+1µM Input DNA Input phoon and EthBr-stained gel images showing dye

Prohead release by wt and acq terminases alone. (D, ii) EthBr- stained agarose gel showing packaging of 5 kb DNA 280 bp 5 kb in the absence and presence of 1 μM YOYO-1 by wt DNA and acq terminases.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1214318109 Dixit et al. Downloaded by guest on October 1, 2021 A B

96(ac) 473

DtsN

N 546 249(q) 1 StsG 562 N10 ts Y terminase packaging 267(N) translocation ts mutations 573 and suppressors EsupK C610 267(N) 249(q)96(ac) 584 H H DsupN L 364 473DtsN 292 300 L 308 C562 281 McsI HcsY E ItsFsup DcsE McsIsup W V D IDresistant portal F,Y F A IDsensitive SS H- L Y- Y Y QQ-

Fig. 5. T4 terminase ID-resistant mutations and terminase-portal interacting translocation functions. (A) ac96, q249, and N267 sites linearly align with each other and with portal clip interacting residue 473 (circled in the gp17 monomer structure) and show complex interdependencies in ID resistance and ter- BIOCHEMISTRY minase function. Various interdependent (shown by arrows) amino acid substitutions at residues 96(ac), 249(q), and 267(N) in the terminase leading to either ID resistance (above line), sensitivity (below line), or loss of terminase function (–) are shown together with the 3D structure. (B) Packaging translocation ts and cs mutations and intra- and intergenic suppressors of the packaging motor proteins. Terminase ts mutations are marked in black, and portal cs mutations are marked in red. Intragenic (18) suppressors are shown with blue arrows, and intergenic (18–21) suppressors are shown by red arrows. Only the portal clip region and translocation channel are shown.

generate forces as high as ∼60 pN. Also, structural studies of the at these sites to be compatible with coupled association with the DNA–YOYO-1 and DNA–TOTO complexes shows that this translocating DNA. Interestingly, although the ac, q,andN sites structure is more consistent with B than A form DNA (A form is are not located in known terminase helicase-like and ATPase 33% compressed compared with B), thus possibly accounting for motifs, they are linearly aligned in the 3D structure, with ac and N comparable packaging inhibitory effects of 9AA and the more separated by about 35 Å or one turn of the helix. Although less strongly binding YOYO-1 in our experiments (21, 22). Overall, direct explanations of the behaviors of mutations in these sites in our direct FRET measurements showing likely DNA compres- the terminase are possible, there is no other direct or conflicting sion in the stalled Y-DNA stem and these results showing ID structural evidence for terminase–DNA translocation contact removal during translocation by the powerful motor appear to be points made in either the T4 or other terminases. One structure of consistent and confirmatory (8, 10). a terminase docked portal has been proposed (4) that does not Mutations can lead to a terminase with enhanced ID resistance apparently fit to this alignment, but it is quite possible that other to packaging inhibition by several IDs. Our experiments suggest docked structures are compatible with the high-resolution gp17 that acq terminase is more able to release dyes from the dye–DNA crystal structure; for example, one is shown aligning the ac, q, N, complex during packaging and hence packages more DNA com- and exposed clip portal channel that apparently interacts with pared with the wt terminase, which could be attributed to the terminase residue 473 (Fig. 5 A and B). There is ample evidence higher binding affinity of this mutant terminase to the dsDNA. It including direct FRET measurements (10) that suggest that this has been found that in bacteriophage Φ29, the contact of packaging terminase-portal structure is highly mobile and may be different in ATPase to ATP or γ-S-ATP resulted in conformational change to the initiation and stalled Y-DNA complex conformations. It is a higher binding affinity toward dsDNA (23). Although ATP hy- tempting to speculate that the DNA duplex is gripped differently drolysis is not required to release some dye from the dsDNAs, the at two positions during translocation: at one point the phospho- presence of ATP or ATP-γ-S does enhance the efficiency of dye diester backbone could be gripped by the highly basic peptide (7 of removal by the terminase, suggesting that the acq mutations might 21 residues are basic surrounding residue 96) and, at the second, be causing a similar, possibly active translocation-like conforma- by insertion or other hydrophobic interaction with the stacked tional change in the terminases, making them more ID resistant. bases in a region of about one helical turn away near to residues We have shown by SDM that rare dye-resistant terminase muta- F249 and W267 (Fig. 5A). The comparable partial removal of dyes tions affecting DNA translocation are located in the N-terminal by the wt and acq mutant terminases alone from the DNA dye ATPase domain. In bacteriophage lambda, two translocation de- complex is likely by the ATP hydrolysis independent gripping and fective mutants (K84A and Y46F) in the ATPase domain were release of the DNA by these terminases. More efficient removal of also characterized (24). The T4 ID-resistant sites have been shown dye by the acq mutant during packaging suggests tighter gripping by SDM to have a high degree of functional coordination despite of the dsDNA between these two regions of the terminase, which being separated by substantial 3D distances in the gp17 3D crystal might help to explain how high force can be generated during structure (Fig. 5A). In view of their interdependence and the as- ATP-generated terminase conformational change and how inter- sociation of these three ID-resistance sites with enhanced removal actions at these two sites might be highly coordinated. of IDs from the translocated DNA, the simplest explanation is that Several terminase ts mutations that block initiation of DNA these mutant sites are located near to points of contact made packaging when grown at high temperature are found to be lo- between the terminase and the translocating DNA. Likely co- cated in its N-terminal portion—here, a mutation in the hinge ordinated binding of DNA near to these sites requires amino acids region of the terminase is able to suppress a portal cs (csD281E)

Dixit et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 mutation that also blocks packaging initiation (Fig. 5B) (reviewed IDs. EthBr and 9AA powder were from Sigma and YOYO-1 from Molecular in ref. 16). A different phenotype is associated with several ter- Probes (Invitrogen). minase ts mutations near its C terminus: accumulation of partially DNA filled proheads (16). Such mutants are thus DNA trans- Mutagensis. Hydroxylamine mutagenesis (26), spontaneous mutagenesis, and location defective, similar to the ID effects seen on translocation. SDM of the gp17 gene was carried out as described in SI Mutagenesis Procedure. In addition to local intragenic terminase suppressors of these C- terminal ts mutations that we have recently isolated—for example, YOYO-1 Staining of the dsDNAs. The double-stranded linear DNAs used in this D584N suppresses tsS546G, and E573K suppresses ts deletion study are 5-kb-length Pl16 DNA linearized by digesting it at the unique PstI site 562Y—a portal cs mutation csM308I located in the clip in- (6), a 280 bp gene20 PCR product, and a 70 bp DNA prepared using oligonu- teraction region suppresses 17 tsD473N (Fig. 5 A and B). It has cleotides synthesized from Sigma. In all of the experiments, the estimated been shown by FRET that the terminase C terminus is located ∼57 staining ratio was roughly 1/20–1/5 dye molecules per DNA base pair. Termi- Å from the portal and approaches the portal by about 6 Å in the nase–DNA ratio was constant, with ∼3 gp17/100 bp of DNA in most of the stalled Y-DNA substrate (10). Taken together, these observations experiments, except for concentration-dependent experiments where differ- suggest that the portal plays a significant role in DNA trans- ent ratios of terminase–DNA were used, as shown in Fig. 3C and Fig. S2. The location that is apparently coupled to interaction with the termi- concentrations of DNA and YOYO-1 were determined spectrophotometrically using the extinction coefficient, є = 6,600 M–l·cm–1 for DNA bases and є = nase C-terminal nuclease domain. The apparent closer approach –l –1 of the terminase to the portal during packaging that is seen in the 96,100 M ·cm for YOYO-1. All samples had a final total volume of 16 μLin2× packaging buffer (10) with 10 mM ATP/ ATP-γ-S or no ATP and were prepared stalled Y-DNA, coupled with a grip-and-release of the DNA by μ the portal, may account for a transient DNA compression that is by adding 1 L of DNA stock to the dye diluted in the buffer, and subsequently incubating at 50 °C for 2 h to achieve homogeneous staining of all molecules connected to the power stroke of the motor complex and to re- — moval of IDs from dsDNA. (13). To make sure the sample was equilibrated that is, that it only gave one band in electrophoresis—the samples were always run on 1.5% (wt/vol) aga- fi Materials and Methods rose and at a 100 V constant eld electrophoresis. Western blotting was carried out using a gp17 antiserum as previously described (27) after running the Bacteriophages, Bacterial Strains, and Growth of Phage Infected Bacteria. T4D samples on 5% native PAGE gel under cold conditions. phage empty large proheads were prepared for packaging in vitro by centrifugation and column chromatography as previously described (6). Cloning, Expression, and Purification of ac and acq Terminases. The ac and acq Escherichia coli DH10B was used for transformation of plasmids in cloning as gp 17 genes were cloned as the wt gp 17 gene in pTYB2 plasmids and were well as for SDM and hydroxylamine mutagenesis experiments. BL21 (DE3) expressed in BL21 (DE3) pLysS, successfully making the gp17-intein/chitin- pLys was used for expression of recombinant proteins. Thirteen different E. binding domain fusion (27). Full-length active ac and acq gp17 was purified coli suppressor strains that insert known different amino acids at amber based on the Impact system of cloning (New England Biolabs, Inc.). codons were used to characterize terminase SDM amber mutant sites (25). Luria–Bertani (LB) medium was used for growth of cells, except for phage DNA Packaging Assays and Analyses. DNA packaging, analysis of gels on Ty- infection experiments in which M9S/20% LB medium (12) was used. Phage phoon imager, and FCS measurements on a Picoquant microtime system were infection in the presence of 9AA was carried out at 30 °C, where the bacteria carried out as previously described (8, 10). were grown at 37 °C and shifted down to 30 °C immediately before infection. The 9AA (3 μg/mL) was added to the culture 30 s after the first in- ACKNOWLEDGMENTS. A set of 13 amber suppressor strains was provided by fection. After 9AA treatment, heads were prepared by glycerol gradient fi J. H. Miller. The authors thank Dr. J. R. Lakowicz and the Center of Fluores- centrifugation similar to the puri cation of proheads described in ref. 6, but cence Spectroscopy for access to the facility in performing the fluorescence fi no column puri cation was done. Plating was carried out on plates containing measurements. We thank Julie Thomas for helpful comments. A.B.D. and 30 mL of Bottom agar (with or without 9AA and EthBr at 1 μg/mL) to deter- L.W.B. were supported by National Institutes of Health (NIH) Grant mine acridine resistance and sensitivity (12). AI11676. K.R. was supported by NIH Grant AI087968.

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