Characterization of nuclease-dependent functions of Exo1p in Saccharomyces cerevisiae.
Exo1p is a member of the Rad2p family of structure-specific nucleases that contain conserved N and I nuclease domains. Exo1p has been implicated in numerous DNA metabolic processes, such as recombination, double-strand break repair and DNA mismatch repair (MMR). In this report, we describe in vitro and in vivo characterization of full-length wild-type and mutant forms of Exo1p. Herein, we demonstrate that full-length yeast Exo1p possesses an intrinsic 5'-3' exonuclease activity as reported previously, but also possesses a flap-endonuclease activity. Our study indicates that Exo1p shares similar, but not identical structure-function relationships to other characterized members of the Rad2p family in the N and I nuclease domains. The two exo1p mutants we examined, showed deficiencies for both double-stranded DNA (dsDNA) 5'-3' exonuclease and flap-endonuclease activities. Examining the genetic interaction of these two exo1 mutations with rad27Delta suggest that the Exo1p flap-endonuclease activity and not the dsDNA 5'-3' exonuclease is redundant to Rad27p for viability. In addition, our in vivo results also indicate that many exo1Delta phenotypes are dependent on the complete catalytic activities of Exo1p. Finally, our findings plus those of other investigators suggest that Exo1p functions both in a catalytic and a structural capacity during DNA MMR.
Tran
Phuoc T
PT
Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR 97201, USA.
Erdeniz
Naz
N
Dudley
Sandra
S
Liskay
R Michael
RM
D016367:Baculoviridae / Q000235:genetics; D020137:Base Pair Mismatch / Q000235:genetics*; D002172:Canavanine / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair / Q000235:genetics*; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D025141:Drug Resistance, Fungal / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000502:physiology*; D045585:Flap Endonucleases; D005656:Fungal Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D005799:Genes, Dominant; D066298:In Vitro Techniques; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017384:Sequence Deletion; D013379:Substrate Specificity; D013489:Suppression, Genetic
D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; C073998:RAD2 protein, S cerevisiae; D002172:Canavanine; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; C028450:exodeoxyribonuclease I