DNA repair in Dictyostelium.
Recent approaches to the study of DNA repair in Dictyostelium discoideum are reviewed. Thymidine auxotrophs facilitate the uptake of labeled thymidine into DNA during its replication and repair. The tmpA600 mutation leads to a loss of thymidylate synthase activity, and tdrA600 results in increased transport of thymidine into the cell. In the HPS401 double mutant (tmpA600tdrA600), thymidine is taken up uniformly into the nuclear and mitochondrial DNAs at levels up to 50-fold that in the wild type. tmpA maps on linkage group III. tdrA is on IV or VI, which cosegregate in strains containing this mutation. Alkaline sucrose gradients of nuclei from HPS401 pulsed for 15 min with [3H]thymidine in axenic medium show that the initially labeled single-strand DNA is about 7 x 10(6) daltons, which may be the size of the replicon. This nascent DNA matures in about 45 minutes to 2 x 10(8) daltons. Ultraviolet light (254 nm) decreases the size of the nascent DNA and delays its maturation. In addition to studies of DNA repair utilizing repair-proficient and -deficient mutants of thymidine auxotrophs, we are currently using two approaches for cloning genes involved in repair: 1) genes are sought that can functionally complement repair defects in Saccharomyces cerevisiae following transformation with a D. discoideum DNA library in YEp 24(URA); 4-NQO is used for the selection of RAD transformants; and 2) we have characterized and purified to near-homogeneity two repair enzymes from D. discoideum--uracil-DNA glycosylase and AP-endonuclease. An N-terminal sequence has been determined for the glycosylase, and a synthetic oligonucleotide probe derived from this sequence will be used to screen for this gene. A similar approach is in progress for the AP-endonuclease.
Deering
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Department of Molecular and Cell Biology, Pennsylvania State University, Althouse Laboratory, University Park 16802.
D004260:DNA Repair; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004023:Dictyostelium / Q000235:genetics* / Q000254:growth & development; D007700:Kinetics; D009154:Mutation; D013936:Thymidine / Q000378:metabolism
D004271:DNA, Fungal; D013936:Thymidine