Involvement of two endonuclease III homologs in the base excision repair pathway for the processing of DNA alkylation damage in Saccharomyces cerevisiae.
DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases. The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps. We have used a genetic approach in Saccharomyces cerevisiae to determine whether or not AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome. We previously reported that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by a model DNA alkylating agent methyl methanesulfonate (MMS) and that this sensitivity can be reduced by deleting the MAG1 3-methyladenine DNA glycosylase gene. Here we report that in the absence of the AP endonucleases, deletion of two Escherichia coli endonuclease III homologs, NTG1 and NTG2, partially suppresses MMS-induced killing, which indicates that the AP lyase products are deleterious unless they are further processed by an AP endonuclease. The severe MMS sensitivity seen in AP endonuclease deficient strains can also be rescued by treatment of cells with the AP lyase inhibitor methoxyamine, which suggests that the product of AP lyase action on an AP site is indeed an extremely toxic lesion. In addition to the AP endonuclease interactions, deletion of NTG1 and NTG2 enhances the mag1 mutant sensitivity to MMS, whereas overexpression of MAG1 in either the ntg1 or ntg2 mutant severely affects cell growth. These results help to delineate alkylation base lesion flow within the BER pathway.
Hanna
Michelle
M
Department of Microbiology and Immunology, University of Saskatchewan, 107 Wiggins Road, SK, S7N 5E5, Saskatoon, Canada.
Chow
Barbara L
BL
Morey
Natalie J
NJ
Jinks-Robertson
Sue
S
Doetsch
Paul W
PW
Xiao
Wei
W
D000478:Alkylation; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001080:Apurinic Acid / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D045643:DNA Repair Enzymes; D004261:DNA Replication / Q000187:drug effects; D004271:DNA, Fungal / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D017353:Gene Deletion; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D011119:Polynucleotides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D018906:Antineoplastic Agents, Alkylating; D004271:DNA, Fungal; D011119:Polynucleotides; D029701:Saccharomyces cerevisiae Proteins; C001790:apyrimidinic acid; D001080:Apurinic Acid; D008741:Methyl Methanesulfonate; D004706:Endodeoxyribonucleases; C083274:Apn1 protein, S cerevisiae; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C445668:MAG1 protein, S cerevisiae; C468953:APN2 protein, S cerevisiae; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C101825:NTG1 protein, S cerevisiae; C116827:NTG2 protein, S cerevisiae; D045643:DNA Repair Enzymes