hSMUG1 can functionally compensate for Ung1 in the yeast Saccharomyces cerevisiae.
There are at least four distinct families of enzymes that recognize and remove uracil from DNA. Family-3 (SMUG1) enzymes have recently been identified and have a preference for uracil in single-stranded DNA when assayed in vitro. Here we investigate the in vivo function of SMUG1 using the yeast Saccharomyces cerevisiae as a model system. These organisms lack a SMUG1 homologue and use a single enzyme, Ung1 to carry out uracil-repair. When a wild-type strain is treated with antifolate agents to induce uracil misincorporation into DNA, S-phase arrest and cellular toxicity occurs. The arrest is characteristic of checkpoint activation due to single-strand breaks caused by continuous uracil removal and self-defeating DNA repair. When uracil-DNA glycosylase is deleted (deltaung1), cells continue through S-phase and arrest at G(2)/M, presumably due to the effects of stable uracil misincorporation in DNA. Pulsed field gel electrophoresis (PFGE) demonstrates that cells are able to complete DNA replication with uracil-substituted DNA and do not experience the extensive strand breakage attributed to uracil-DNA glycosylase-mediated repair. As a result, these cells experience early protection from antifolate-induced cytotoxicity. When either UNG1 or SMUG1 functions are reintroduced back into the null strain and then subjected to antifolate treatment, the cells revert back to the wild-type phenotype as shown by a restored sensitivity to drug and S-phase arrest. The arrest is accompanied by the accumulation of replication intermediates as determined by PFGE. Collectively, these data indicate that SMUG1 can act as a functional homolog of the family-1 uracil-DNA glycosylase enzymes.
Elateri
Imane
I
Department of Molecular Biology, University of Medicine and Dentistry of NJ-SOM and GSBS, 2 Medical Center Drive, Stratford, NJ 08084, USA.
Tinkelenberg
Beverly A
BA
Hansbury
Michael
M
Caradonna
Sal
S
Muller-Weeks
Susan
S
Ladner
Robert D
RD
D000818:Animals; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases; D004261:DNA Replication / Q000235:genetics / Q000502:physiology; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009699:N-Glycosyl Hydrolases / Q000235:genetics* / Q000378:metabolism*; D030781:Organisms, Genetically Modified; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase
D014498:Uracil; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C498095:SMUG1 protein, human; D051981:Uracil-DNA Glycosidase