Functional specialization of Chlamydomonas reinhardtii cytosolic thioredoxin h1 in the response to alkylation-induced DNA damage.
DNA damage occurs as a by-product of intrinsic cellular processes, like DNA replication, or as a consequence of exposure to genotoxic agents. Organisms have evolved multiple mechanisms to avoid, tolerate, or repair DNA lesions. To gain insight into these processes, we have isolated mutants hypersensitive to DNA-damaging agents in the green alga Chlamydomonas reinhardtii. One mutant, Ble-1, showed decreased survival when it was treated with methyl methanesulfonate (MMS), bleomycin, or hydrogen peroxide (H2O2) but behaved like the wild type when it was exposed to UVC irradiation. Ble-1 carries an extensive chromosomal deletion that includes the gene encoding cytosolic thioredoxin h1 (Trxh1). Transformation of Ble-1 with a wild-type copy of Trxh1 fully corrected the MMS hypersensitivity and partly restored the tolerance to bleomycin. Trxh1 also complemented a defect in the repair of MMS-induced DNA strand breaks and alkali-labile sites. In addition, a Trxh1-beta-glucuronidase fusion protein translocated to the nucleus in response to treatment with MMS. However, somewhat surprisingly, Trxh1 failed to correct the Ble-1 hypersensitivity to H2O2. Moreover, Trxh1 suppression by RNA interference in a wild-type strain resulted in enhanced sensitivity to MMS and DNA repair defects but no increased cytotoxicity to H2O2. Thioredoxins have been implicated in oxidative-stress responses in many organisms. Yet our results indicate a specific role of Chlamydomonas Trxh1 in the repair of MMS-induced DNA damage, whereas it is dispensable for the response to H2O2. These observations also suggest functional specialization among cytosolic thioredoxins since another Chlamydomonas isoform (Trxh2) does not compensate for the lack of Trxh1.
Sarkar
Nandita
N
School of Biological Sciences and Plant Science Initiative, University Of Nebraska-Lincoln, Lincoln, NE 68588-0666, USA.
Lemaire
Stéphane
S
Wu-Scharf
Danxia
D
Issakidis-Bourguet
Emmanuelle
E
Cerutti
Heriberto
H
D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002470:Cell Survival; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D004247:DNA / Q000187:drug effects; D004249:DNA Damage; D004260:DNA Repair; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009153:Mutagens / Q000494:pharmacology; D019697:Onions / Q000166:cytology / Q000502:physiology; D010641:Phenotype; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
D020418:Algal Proteins; D029968:Escherichia coli Proteins; D009153:Mutagens; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C491202:trxC protein, E coli; D013879:Thioredoxins; D004247:DNA; D008741:Methyl Methanesulfonate