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

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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.
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