Barbara W. Durkacz

The fission yeast cell cycle control gene cdc2: isolation of a sequence suc1 that suppresses cdc2 mutant function.

SummaryA DNA fragment called suc1 has been found to rescue cells mutated in the cell cycle control gene cdc2 of the fission yeast Schizosaccharomyces pombe. The suppressing activity of suc1 is observed when it is present on a multicopy number plasmid. The gene does not hybridise to cdc2 and maps elsewhere in the genome. Its effect is cdc2 allele specific suggesting that it interacts directly with the cdc2 gene function.

Cloning, sequencing and transcriptional control of the Schizosaccharomyces pombe cdc10 'start' gene.

The cdc10 ‘start’ gene from the fission yeast Schizosaccharomyces pombe has been cloned by rescue of mutant function. It is present as a single copy in the haploid genome. Hybridisation of the gene to Northern blots has identified a low abundance 2.7‐kb polyadenylated RNA. Study of RNA extracted from cells both entering stationary phase and undergoing synchronous cell divisions suggests that commitment to the cell cycle is not controlled by regulation of cdc10 transcript level. DNA sequence analysis of the gene has identified an open reading frame capable of encoding a protein of mol. wt. 85 400. The putative cdc10 gene product shows no significant primary structure similarity with products of other fission and budding yeast cell cycle genes, or with other protein sequences in several databases.

Inhibition of (ADP-ribose)n biosynthesis retards DNA repair but does not inhibit DNA repair synthesis

Abstract Inhibition of ADP-ribosyl transferase activity by 3-aminobenzamide results in an inhibition of DNA excision repair. We have assayed repair synthesis in L1210 mouse lymphoblastoid cells following treatment with dimethyl sulphate, or exposure to γ-rays, in the presence and absence of 3-aminobenzamide. Repair synthesis increases with increasing DMS dose, and this increase is enhanced in the presence of 3-aminobenzamide at higher doses of dimethyl sulphate. The rate of increase of repair synthesis with increasing γ-irradiation is the same in 3-aminobenzamide-treated as in control cultures. Thus, the inhibition of DNA strand-rejoining, when cells are treated with 3-aminobenzamide, is not due to an inhibition of repair synthesis.

Transcription of the cdc2 cell cycle control gene of the fission yeast Schizosaccharomyces pombe

The cdc2 gene plays a central role in the control of the mitotic cell cycle of the fission yeast Schizosaccharomyces pombe. It is required in G1 at start for commitment to the mitotic cycle and then again in G2 where it determines the timing of mitosis. We have identified the cdc2 gene transcript as a 1.6‐kb polyadenylated mRNA. This transcript is generated after four introns have been spliced out; there is no evidence for differential splicing. The level of cdc2 transcript does not change during a shift between cell proliferation and stationary phase or during the mitotic cell cycle. Overproduction of the cdc2 transcript does not alter the normal cell cycle. We conclude that the cell cycle is not controlled by changes in either the cdc2 transcript level or in its processing. A gene adjacent to cdc2 called cdc2L has also been identified. This encodes three transcripts of 1.0–1.3 kb in length, at least two of which are cell cycle regulated. Their levels peak during S‐phase and are increased in certain cell cycle mutants. This gene may code for a product which is required for the mitotic cell cycle.

Inhibitors of nuclear ADP-ribosyl transferase retard DNA repair after N-methyl-N-nitroso-urea: Further evidence for the involvement of (ADP-ribose)n in DNA repair

ADP-ribosyl transferase is a nuclear enzyme that transfers ADP-ribosyl residues from NAD ÷ to chromatin proteins, to form mono-, oligoand poly(ADPribose) (reviews [1-4]) . The enzyme is totally dependent on DNA [5] and is markedly stimulated by fragmentation of the DNA [6-9]. Evidence has accumulated over the last 5 years that (ADP-ribose)n participates in the cellular recovery from DNA damage [10,11 ]. Both radiation and alkylating agents lower the cellular NAD content [6,12-14] and thereby interfere with glycolysis. This drop in cellular NAD is mediated by nuclear ADP-ribosyl transferase [10,12]. Poly~ADP-ribose) in intact cells is increased after DNA damage [15]. The rejoining of DNA strand breaks induced by exposure to dimethyl sulphate is retarded by representatives of all 4 classes of inhibitors of ADP-ribosyl transferase [ 10,t 1 ]. Furthermore, these same enzyme inhibitors also potentiate the cytotoxicity of dimethyl sulphate [10,11,1 6]. Finally, lowering the cellular NAD level by nutritional deprivation of nicotinamide totally prevents DNA repair after dimethyl sulphate [10]. All this evidence argues forcefully that (ADPribose)n participates in DNA repair. However, the above experiments examined repair of damage induced by dimethyl sulphate. It is known that the spectrum of damage induced in DNA depends

Segregation kinetics of colicinogenic factor col E1 from a bacterial population temperature sensitive for DNA polymerase I.

SummaryWe have studied the segregation kinetics of the bacterial plasmid ColE1 from a population of cells temperature sensitive for DNA polymerase I. The results indicate that there are about twelve plasmid copies per cell of the strain used and that segregation is probably random.

The fission yeast cell cycle control gene cdc2: structure of the cdc2 region

SummaryThe cdc2 cell cycle control gene of Schizosaccharomyces pombe has been identified on a 3 kb DNA fragment. The gene is unique in the genome and is located near to a 5S ribosomal RNA gene. When a plasmid containing DNA sequences adjacent to the cdc2 gene is transformed into certain temperature sensitive cdc2 mutants it allows colony formation at the restrictive temperature. This was shown to be due to the plasmid interacting with the cdc2 chromosomal region and picking up the temperature sensitive allele of the cdc2 gene. Over expression of these temperature sensitive alleles presumably leads to sufficient activity of the thermolabile product to allow normal cdc2 function. In this way two cdc2 alleles have been cloned.

A mammalian cell variant in which 3-aminobenzamide does not potentiate the cytotoxicity of dimethyl sulphate

Variants of mouse leukaemia L1210 cells have been isolated in which cytotoxicity to dimethyl sulphate is not fully potentiated by ADP-ribosyl transferase inhibitor 3-aminobenzamide, as occurs in normal L1210 cells. These variants were selected after mutagenesis by growing the cells in dimethyl sulphate and 3-aminobenzamide. The characterisation of one of these variants is described. Variant 3 cells repair low doses of DNA damage in the presence of ADP-ribosyl transferase inhibitors. The Vmax of the ADP-ribosyl transferase enzyme in these cells is only increased 35% compared to normal wild-type L1210 cells. The basal DNA ligase I activity is increased 66% above wild-type whereas DNA ligase II activity appears to be unchanged. The most striking observation, however, is that the DNA ligase II activity is not increased after dimethyl sulphate treatment as occurs in wild-type L1210 cells. It seems that by increasing DNA ligase I levels these cells can survive DNA damage in the presence of 3-aminobenzamide. This variant (mutant) provides genetic evidence for our previously published hypothesis that (ADP-ribose)n biosynthesis is required for efficient DNA repair after DNA damage by monofunctional alkylating agents, because ADP-ribosyl transferase activity regulates DNA ligase activity. This variant is the first mammalian cell reported in which DNA ligase activity is altered, as far as we are aware. In yeast, a DNA ligase mutant has a cell division cycle (cdc) phenotype. Presumably, DNA ligase is essential for DNA synthesis, repair and recombination. The present variant provides further evidence that in mammalian cells, DNA ligase II activity is related to ADP-ribosyl transferase activity.

(ADP-Ribose)n, A New Component in DNA Repair

It has beeri,known for about 25 years that DNA-damaging chemicals and radiation will lower cellular NAD levels. We provide a biochemical explanation for these observations which leads us into the hypothesis that NAD metabolism participates in DNA repair.

Isolation of a DNA Ligase Mutant from L1210 Cells

Current evidence suggests that the ADP-ribosylation of chromatin proteins is involved in DNA repair [2]. Much of this evidence comes from the use of inhibitors of ADPRT, such as 3-aminobenzamide (3AB), which retards DNA strand rejoining following DNA damage and potentiates the cytotoxicity of DNA damaging agents. Alkylation damage increases DNA ligase activity, predominantly that of DNA ligase II. This increase is prevented by ADPRT inhibitors. We have previously suggested that the requirement for ADPRT in DNA repair is at the ligation step [1].