Photochemistry and Photobiology, 1999, zyxwvuts 70(4): zyxwvut 505-51 1 Symposium-in-Print Mutation Spectrum Induced by Singlet Oxygen in zyx Escherichia coli Deficient in Exonuclease 111 Lucymara F. Agnez-Limatl, Paolo Di Mascio2,Rita L. Napolitano3, Robert P. Fuchs3and Carlos F. M. Menck*’ ‘Departamento de Microbiologia, lnstituto de CiQncias Biomedicas, Universidade de zyxwv Slo Paulo, Slo Paulo, Brazil; *Departamento de Bioquimica, lnstituto de Quimica, Universidade de Slo Paulo, Slo Paulo, Brazil and 3Ecole Superieure de Biotechnologie de Strasbourg, Strasbourg, France Received 1 March 1999; accepted 24 June 1999 ABSTRACT zyxwvutsr The repair of singlet oxygen ( ‘02)-induced DNA lesions zyxwvu requires several enzymes of the nucleotideand base excision repair pathways, including exonuclease III and endonucle- ase IV that are known apurinidapyrimidinic-endonucleases in Escherichia coli. In order to better understand the rel- evance of exonuclease 111 on the repair of these lesions, we investigated the mutagenic events that result from the replication of a ‘O,-damaged plasmid in an exonuclease- deficient host (xth). The mutation spectrum in the tRNA supF gene target indicated that the absence of exonucle- ase I11 does not change the types of mutations induced by *02 (mostly of G:C + T:A and G:C + C:G transver- sions). However, the spectrum shows that the mutations are scattered in the supF gene, which is significatively different from the one obtained in wild-type bacteria. Thus, exonuclease I11 may act on the repair of ‘O,-in- duced lesions altering the DNA repair sequence specific- ity. INTRODUCTION The electronic excited molecular oxygen (singlet oxygen, lo2$) is known to be a product of several biological pro- cesses, including (I) defense mechanisms of living organ- isms such as in phagocytosis (1); (2) hormonal activity of prostaglandins; (3) photochemotherapy utilizing the photo- dynamic action of synthetic dyes that can transfer an electron *To whom correspondence should be addressed at: Departamento de Microbiologia. 1CB2, USP. Av. Prof. Lineu Prestes, 1374, SHo Paulo, 05508-900, SP, Brazil. Fax: 55.1 1.818.7354; e-mail: cfmmenck@usp.br ?Current address: Departamento de Biologia Celular e GenCtica, Centro de Biocisncias, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil. $Abbreviations: AP, apurinic/apyrimidinic; dGuo, 2’-deoxyguano- sine; 8-oxodGuo, 8-0~0-7,8-dihydro-2’-deoxyguanosine; 4-OH-8- oxodGuo, 4-hydroxy-8-oxo-4,8-dihydro-2’-deoxyguanosine; FPG, formamidopyrimidine-DNA-N-glycosylase; NDP02, 3.3’-( 1.4- naphthylidene) diproprionate endoperoxide; ‘02, singlet oxygen. zyxwvuts 0 1999 American Society for Photobiology 003 1-8655/99 $S.OO+O.OO to a substrate leading to the formation of a substrate radical (type I reaction), or alternatively the energy can be trans- ferred to oxygen yielding lo2 (type II reaction); (4) clinical manifestations of toxic agents like psoralens and (5) inborn errors of metabolism exemplified by erythropoietic porphyr- ia; (6) lo2 can also be generated in a dark reaction (chemiex- citation), for example, lo2 can be generated enzymatically by the action of peroxidases or lipoxygenases or by the re- action of hydrogen peroxide with hypochlorite or peroxyni- trite (2) or by thermodecomposition of dioxetanes (3,4). This reactive species can damage cellular macromolecules like DNA, and these DNA lesions are processed within the cells by DNA replication and repair machineries. This may result in cell killing or mutations that are both the basis of several human degenerative diseases, such as aging and cancer (5,6). The DNA damage induced by lo2 is mainly at guanine sites and several DNA alterations have been identified when free 2’-deoxyguanosine (dGuo) or DNA is treated with lo2 produced by methylene blue in the presence of light (7-1 1). This reaction leads to the formation of two main products: the 4R* and 4S* diasteromers of 4-hydroxy-8-oxo-4,8-di- hydro-2’-deoxyguanosine (4-OH-8-oxodGuo) and 8-oxo- 7,8-dihydro-2’-deoxyguanosine (8-oxodGuo) (Fig. 1) (7- ll), but other mutagenic andor lethal lesions could be formed. Concerning the mutagenicity, 8-oxodGuo is known to mispair with 2‘-deoxyadenosine yielding G to T transver- sions (12,13). Many DNA repair pathways have been characterized as implicated in the removal zyxw of DNA damage induced by lo2. The formamidopyrimidine-DNA-N-glycosylase (FPG) is probably the most important protein related to oxidative DNA damage: this enzyme excises 8-oxodGu0, when form- ing a pair with cytosine and also recognizes many imidazole ring-opened purines (14,15). After removing the altered base by its glycosylase activity, it cleaves the phosphodiester bound at the abasic site formed, acting as an apurinic/apyr- imidinic (AP)-lyase (16-18). The UvrABC system partici- pation in the repair of DNA oxidative lesions was suggested after the analysis of the transformation efficiency of plas- mids treated with methylene blue plus light. It was observed that plasmid inactivation was more pronounced in bacteria 505