Differential chromosomal and mitochondrial DNA synthesis in temperature-sensitive mutants ofUstilago maydis

Abstract

The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutants of the smut fungus Ustilago maydis after incubation at the restrictive temperature (32 degrees C) for eight hours. Mutants ts-220, ts-207, ts-432 and ts-346 were found to have an overall reduction in the synthesis of both nuclear and mitochondrial DNA comparison to the wild-type. In mutants ts-20, tsd 1-1, ts-84 and pol 1-1 nuclear DNA synthesis was depressed relative to mitochondrial synthesis. The DNA-polymerase mutant pol 1-1 had persistent nuclear synthesis at about 50% of the rate of synthesis of mitochondrial DNA and similar behavior was observed in a diploid homozygous strain. Mutant ts-84 had an initial burst of DNA synthesis which was reduced for nuclear but not mitochondrial synthesis after three hours preincubation at 32 degrees C. tsd 1-1 and ts-20 had nuclear residual synthesis amounting to about 25% of the relative rate of mitochondrial synthesis which correlates to increasing UV sensitivity of these strains on incubation at 32 degrees C. A pol 1-1 ts-84 double mutant had an additive loss of nuclear DNA synthesis which indicates that the steps of replication involved may be sequential.

Some Aspects of Eukaryotic DNA Replication

“…At the nonpermissive temperature, the latter cells synthesize amounts of DNA that are additive with respect to the parents carrying single mutations. Kinetic studies of DNA synthesis and development of sensitivity to ultraviolet (UV) light (402) suggest that the ts -84 gene product acts after pol l-I. Mutants of U. maydis that are sensitive to UV light and are auxotrophic for pyrimidines have also been isolated (425,426).…”

Molecular cloning of a gene required for DNA replication in Ustilago maydis

“…The mutant was observed to have a slight antimutator phenotype (Holliday et al 1978). It was also observed that the defect in DNA synthesis was due to a selective block of nuclear, rather than mitochondrial, DNA replication (Unrau 1977).…”

“…Several hundred mutants, temperature sensitive for growth, were screened for their inability to synthesize DNA at the restrictive temperature. Five of these mutants were found to be blocked in DNA synthesis and were characterized in more detail by both genetic and biochemical means (Unrau andHolliday 1970, 1972). When cell-free extracts prepared from these mutants were assayed directly for DNA polymerase activity, it was found that the extract from one of them, mutant ts92a, had a reduced level of activity, suggesting that the DNA synthesis defect was due to a defective DNA polymerase (Jeggo et al 1973).…”