Saccharomyces cerevisiae RAD27 complements its Escherichia coli homolog in damage repair but not mutation avoidance.
In eukaryotes, the flap endonuclease of Rad27/Fen-1 is thought to play a critical role in lagging-strand DNA replication by removing ribonucleotides present at the 5' ends of Okazaki fragments, and in base excision repair by cleaving a 5' flap structure that may result during base excision repair. Saccharomyces cerevisiae rad27Delta mutants further display a repeat tract instability phenotype and a high rate of forward mutations to canavanine resistance that result from duplications of DNA sequence, indicating a role in mutation avoidance. Two conserved motifs in Rad27/Fen-1 show homology to the 5' --> 3' exonuclease domain of Escherichia coli DNA polymerase I. The strain defective in the 5' --> 3' exonuclease domain in DNA polymerase I shows essentially the same phenotype as the yeast rad27Delta strain. In this study, we expressed the yeast RAD27 gene in an E. coli strain lacking the 5' --> 3' exonuclease domain in DNA polymerase I in order to test whether eukaryotic RAD27/FEN-1 can complement the defect of its bacterial homolog. We found that the yeast Rad27 protein complements sensitivity to methyl methanesulfonate in an E. coli mutant. On the other hand, Rad27 protein did not reduce the high rate of spontaneous mutagenesis in the E. coli tonB gene which results from duplication of DNA. These results indicate that the yeast Rad27 and E. coli 5' --> 3' exonuclease act on the same substrate. We argue that the lack of mutation avoidance of yeast RAD27 in E. coli results from a lack of interaction between the yeast Rad27 protein and the E. coli replication clamp (beta-clamp).
Ohnishi
Gaku
G
Graduate School of Life Sciences, Tohoku University, Sendai, Japan.
Daigaku
Yasukazu
Y
Nagata
Yuki
Y
Ihara
Makoto
M
Yamamoto
Kazuo
K
D000123:Acetyltransferases; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D045585:Flap Endonucleases / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011401:Promoter Regions, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; C489181:tonB protein, E coli; D000123:Acetyltransferases; C107118:ELO2 protein, S cerevisiae; D004256:DNA Polymerase I; D045585:Flap Endonucleases; C468323:RAD27 protein, S cerevisiae