Site-specific mutagenesis by a propanodeoxyguanosine adduct carried on an M13 genome.

P.C Burcham,L.J Marnett

doi:10.1016/s0021-9258(19)61984-8

Abstract

The spectrum of mutations induced upon in vivo replication of an M13 genome containing a site-specifically located propanodeoxyguanosine (PdG) adduct was determined. PdG was used as a model for the major deoxyguanosine adduct produced on reaction of DNA with the endogenous genotoxin malondialdehyde. PdG was introduced at position 6256 of M13MB102 by ligating the oligodeoxynucleotide 5'-GGT(PdG)TCCG-3' into an 8-base gap in the (-)-strand of duplex M13MB102. Replication of the adducted strand was maximized by incorporation of uracil into the unadducted (+)-strand. Following replication of dG-containing and PdG-containing M13MB102 genomes in Escherichia coli JM105, frameshift mutations were detected as phenotypic changes in the lacZ alpha marker gene. Base pair substitutions were detected by differential hybridization using 32P-labeled 13-mers bearing different bases opposite position 6256. Neither frameshift nor base pair substitution mutations were detected following replication of PdG-adducted genomes in non-SOS-induced JM105. However, PdG-->T transversions and PdG-->A transitions were detected following transformation of PdG-adducted M13MB102 into SOS-induced JM105. Both types of mutations were detected at comparable frequencies, and the total mutation frequency was approximately 2%. The results indicate that PdG is an efficient premutagenic lesion in E. coli strains in which the SOS response is induced.

Highlights

Lication of an M13 genome containinga site- are poorly understood, they are likely related to its ability to located propanodeoxyguanosine (PdG) adduct was de- react with selected deoxynucleosides [8,9,10,11]
PdGwas the reactionof MDAwith deoxynucleosides and/or DNA at neulo8piconal-oiwbttgrriaoaoiornsdtndeeigeoupoongclxfiaecyopdtanfhtuueiiancortlanaetdochpdit(eloiu5d-si)'ceni--ttGstietoodd6rGonG2atfTsh5n-tc6e(rdPoaooundnffntGdaM dai)undwT1pid3anCalPM nuegsCxdcBmGtGM e1a-0d-i3n12xa'3t(inom+M by)li-Bizsge1tadr0ta2ibnn. ygdR.eintFphcloieo-lr--htpervyeaWripdliaeemtpniihcHcdeaoDv[iiNenls,dAr2aeic-ocaafe1t]nre,pWatsultyrts-hpifanayo-tnruildmMn0d(i,hdG3tuoHhmp)ea-uxtaorininsrnetse((soMa1nca3e,ttG,ios1,dni4gFe)nori.gifivfM.iac1Dtai)vnA(te8l,weo9vfi1et.h'glsuRsaiinenncgientlnhee-te, containing M13MB102genomes in Escherichiacoli stranded DNA from Ml3MB102 leads to an increase in lacZ, JM105, frameshift mutations were detected as phenomutations on replication in Escherichia coli JM105 [15]
The role of endogenousgenotoxins in tumorigenesis has from M13MB102, a recombinant M13 phage that contains the emerged as an important research area asa result of the de- mutable sequence of Salmonella typhimurium hisD3052 as an tection of a variety of adducts derived from endogenous elect- in-frame replacement for a portion of the polylinker region of rophiles in human DNA [1,2,3,4]

Summary

Introduction

The spectrum of mutations induced uponin vivo rep- though the mechanisms underlying the mutagenicityof MDA lication of an M13 genome containinga site- are poorly understood, they are likely related to its ability to located propanodeoxyguanosine (PdG) adduct was de- react with selected deoxynucleosides [8,9,10,11]. Neither frameshift nor base pair sub-C + T and A+G transitions, and frameshift mutation(1s5).If stitution mutations were detected following replicationne assumes that all of the mutations were targeted to the site of PdG-adducted genomes in non-SOS-induced JM105. The instabilityof M,G t o the conditions of oligoquencies, and the total mutation frequency was appnruoclxeio-tide deprotectionprecludes construction of viral genomes mately 2%.The results indicate thatPdG is an efficient containing this lesion. To circumvent this problem, we have premutagenic lesion in E. coli strains in which the SOS usedthestructuralanalogue propanodeoxyguanosine $ Present address: Dept. of Clinical and Experimental Pharmacology, To obtain a morecomprehensive mutational spectrum for

Objectives

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Conclusion