Abstract
The adenovirus single-stranded DNA-binding protein (DBP) is an essential factor in viral DNA replication. Three temperature-sensitive (ts) adenoviruses (Ad2+ND1ts23, Ad2ts111A, and Ad5ts125) are known to have single amino acid substitutions in their DBPs that result in defective DNA replication at the nonpermissive temperature. To elucidate the mechanism(s) involved in the ts phenotype, we purified the three mutant DBPs and studied their DNA-binding properties and their ability to support DNA replication in an in vitro system. The results confirm that the three ts DBPs were incapable of supporting DNA replication at the nonpermissive temperature (40 degrees C). The defect was found at both the initiation and elongation steps of DNA replication. The 2-fold stimulation of pTP.dCMP formation by the DBP was lost by prior heating of the ts DBPs. The pronounced effect of the DBP on the early elongation process was severely diminished, but not abolished, by prior heating to 40 degrees C. The functional change at 40 degrees C was irreversible, as the ts DBPs preincubated at 40 degrees C were no longer active when assayed at 30 degrees C. Upon heating to 40 degrees C, all three ts DBPs lost their ability to bind to oligonucleotides, although they still retained some binding activity for large single-stranded DNAs such as M13 DNA. Thus, the inability of these three ts DBPs to support DNA replication is attributable to their altered DNA-binding properties.
Highlights
From the Department of Virology and Molecular Biology, St
The abbreviations used are: Ad, adenovirus; ss, single-stranded; ds, double-stranded; DBP, 72-kDa adenovirus ssDNA-binding protein; ts, temperature-sensitive; DNA-prot, adenovirus DNA with 55kDa terminal protein covalently linked to each 5' end; precursor to the terminal protein (pTP), 80-kDa precursor to terminal protein;Adpol, adenovirus DNA polymerase; V pTP and V pol, crude extracts prepared from HeLa cells infected with recombinant vaccinia viruses expressing Ad pTP and pol, respectively;NF, HeLa cell nuclear factors involved in adenovirus DNA replication; 2-ME, 2-mercaptoethanol; BSA, bovine serum albumin; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfoniaccid; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis
We subsequently realized that comparisons based on NaCl concentrations were inaccurate because the elution peak for the same DBP varied significantly across column umn [47].The ts DBPs were labeled in vivo with ['"Plorthophosphate runs depending on the age of the column
Summary
Vaccinia viruses expressing Adpol and pTPwere used to infect HeLa tecEnm(1(hdcnia.nny0maeztMgemurnymolrramaa(iile1egttdn)dueer4/diamrsrn7fniecreaBCladdea)lnesili(ofc/-f)5d[mRr,alY0toal[hTamfbmC-udys4~to-imi,hoB/'l~Pmcpy)auer'om]cfsPmetArlthayoiu]oDevtTnms~ilsenNu()PdC4DecDaAf,lT5NruepoR(0loPhA34amP0etob.,i2os0Cdsecen0Spelioamst0/ch-vrmkhoNcCgauiwhmk/lniegse/iaawmeonLn)rsdlatzame)lfs/bEyreMfooowwnrclmoraga)eeram,nnrlorteaeon[dSs:nwIsr;ipCid-sge3lusaN[mNis'D3rnnP,uc2akdB]Nhcn,PdelaiiA~dAs]eotsorsa-me-oTDtrrc[dotc.3PeehNeRdl,oalfl4A(iulergpc3uo,sl-aha5,oltma0rolor-sgso0is~es;case0Np[Hter(i3Chodfeoi]Haasweilnl/et-t-e]eu-cAuwmAcrH7seuoe.nd5iesilclitxnlpLunhhmDsiuemfa,nMectcN1ghancltc0eiAntaepv1eaaltdph-e5drslpllotpnydcreshHolou)eypxeftscotwhet,ofclfrLoiae0erpanaiacra.clb5aesrartsleeosccoppedamrloexlluretunpielitatonlctirtprscfioaeaeaotin,cevVir(nnotxie65,ionctdpcn0)ru0ooogam(s.aannlc82hl,Mnstytt5oa0sapddsi(pi.gnH5ent4(lpsi)i3doEnapcnf)ecsglrPg.soisccRoobiEeetngrftehdeleSthnldaVaea/aeictKftbtrniihatpennyieOcrdoTdeodrtnSHioePucaks3tprg,a,usis5lwnht0niepcaVw.nend1aHsargea5eteapin7sooDpmosb.(fn15p2eleEXo,rt4or5aeuAcgu0fhpnonmpoEuuaemdtMIsn-nrtocepdiiVoannedtoiMflslgf.in/lecpDeumfge(cerTs3lCnBtoogtbe7tPelms)s,Pdy)d,,e,,. TemperatureA-sdDeenNnsoAiPtvi-rvibroeiutnesdininsg amounts of DBP with 50 ng of DNA-prot, 1 pl of V pol, 1 pl of V solution consisting of 10 mM Tris-HCI, pH 8.0, 20 mM NaCI, 1 mM pTP, 0.3 pl of uninfected HeLa cell nuclear extract, 0.8 pg of creatine EDTA, 2 mM 2-ME, 200 pg/ml BSA, and 10%glycerol.The mixtures kinase, 40 mM HEPES/KOH,pH 7.5, 7 mM MgCI2, 1 mM DTT, were kept on ice for a t least 30 min and analyzed at 4 "C by agarose. Pellets were immunostained with an anti-DBPmonoclonal antibody (B6) and an washed once with 5%trichloroacetic acid and dissolved in 40 pl of a anti-mouse IgG HRP conjugate (Bio-Rad) according to themanufacloading solution consisting of 50 mM Tris-HCI, pH 6.8, 1% SDS, 1% turer's protocol. Products were separated by SDS-PAGE [44] and visualized by autoradiography
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