Siv Ljungquist

Altered DNA ligase III activity in the CHO EM9 mutant

Delayed joining of DNA strand breaks and a high spontaneous level of sister-chromatid exchanges (SCEs) are characteristics of the mutant cell strain EM9 of Chinese hamster ovary (CHO) cells. The introduction of the human gene XRCC1 into EM9 cells reverts the phenotypic properties of EM9 to those of the wild type. We have investigated both DNA ligase activities and a protein which stimulates DNA ligase activity in mutant EM9 cells, XRCC1-transfectant H9T3-7-1 cells and wild-type AA8 cells. Our results, which demonstrate both a decreased DNA ligase activity in EM9 cells using poly(rA).oligo(dT) as substrate and a decreased ability of DNA ligase III to form a covalent DNA ligase III-adenylate intermediate with AMP, clearly indicate an altered DNA ligase III activity in the mutant. Furthermore, the AMP-binding capacity of DNA ligase III and its enzymatic activity with the synthetic polymer were restored after transfection of EM9 with the human XRCC1 gene. Immunoblotting data suggest that the XRCC1 gene does not code for DNA ligase III. In conclusion, the data indicate that the EM9 cell strain has an altered DNA ligase III activity that can be restored by the XRCC1 gene product.

Apurinic and Apyrimidinic Sites in DNA

An outline review is given of the chemical properties of apurinic and apyrimidinic sites in DNA, the rate of introduction of such sites under different conditions, secondary lesions such as chain breaks, and the properties of endonucleases that specifically attack DNA at apurinic sites.

Mammalian DNA endonuclease acting at apurinic sites: Absence of associated exonuclease activity

Apurinic (and/or apyrimidinic) sites in DNA occur as a consequence of spontaneous DNA hydrolysis at neutral pH [ 11, after treatment of DNA with alkylating agents [2] or ionizing radiation [3,4] and by the action of enzymes that cleave N-glycosidic bonds in DNA [5]. The apurinic sites are presumably efficiently removed by an excision repair mechanism in vivo and endonucleases that specifically attack DNA at such sites seem to be present in all living cells [6,7]. Yajko and Weiss [8] have recently shown that the E. coli enzyme of this type, endonuclease II [6,9] is identical with E. coli exonuclease III/DNA 3’-phosphatase [ 10,l l] It was further found that the endonuclease II and exonu. clease III activities of HemophiZus influenzae also are associated in the same protein which suggested a more general phenomenon [8,12]. Here it is shown that while the E. coli endonuclease II/exonuclease III/DNA 3’-phosphatase activities are all present in a purified preparation of E. colt’ exonuclease III, in confirmation of the work of Yajko and Weiss [8], the otherwise similar mammalian endonuclease [3,7] does not possess associated exonuclease and phosphatase activities. Further, an enzyme activity similar to E. coli exonuclease III does not appear to be present in mammalian cells.

p53 splice acceptor site mutation and increased HsRAD51 protein expression in Bloom's syndrome GM1492 fibroblasts

GM1492 human diploid skin fibroblasts derived from a patient with Blooms syndrome (BS), lack detectable p53 mRNA and protein as shown by Northern and Western blotting, and express an increased RecA-like activity. Here we demonstrate that the p53 gene is grossly intact in GM1492 cells according to Southern blotting. DNA sequencing did not reveal any mutations in the promoter region of p53. A highly sensitive RT-PCR produced a p53 cDNA fragment that was shorter than expected. DNA sequence analysis of p53 cDNA showed that exon 6 was missing, explaining the shorter PCR product. Furthermore, sequencing of genomic DNA revealed a base substitution at the nucleotide preceding the AG splice acceptor site of intron 5. The omission of exon 6 creates a frameshift at the junction of exons 5 and 7, and a premature stop codon in exon 7. The aberrant transcript is predicted to encode a truncated p53 protein containing 189 amino acid residues. Moreover, Western blotting demonstrated elevated HsRAD51 protein levels in GM1492 cells. The lack of sufficient levels of wild-type p53 and increased levels of HsRad51 protein may contribute to the elevated RecA-like activity in the GM1492 fibroblasts.

RecA-like activity in mammalian cell extracts of different origin

The occurrence of a RecA-like activity similar to the one detected in the fibroblast cell line GM1492 derived from a patient suffering from the autosomal recessive disease Blooms syndrome has been investigated in cell extracts of different origin. The formation of D-loop containing joint molecules from phi X174 RFI DNA and fragments of phi X174 single-stranded DNA by partially purified extracts was measured by a filter binding assay. The RecA-like activity, dependent on ATP and Mg2+, was detected at an elevated level only in the human and rodent cell lines, GM1492 and CHO respectively. The level of activity in DNA-cellulose-purified cell extracts from these cell lines was 4-7-fold higher compared to normal human fibroblasts. Low levels of activity were also detected in extracts from two additional Blooms syndrome fibroblast cell lines, Fanconis anemia fibroblasts, virus- (Epstein-Barr virus, Simian virus 40) transformed human cells and human placenta. Cell extracts from rat testis, spleen and calf thymus were also of low activity.

Reduced DNA ligase activity in etoposide resistant human lymphatic leukaemia CEM cells.

Drug resistance is an obstacle preventing success of cancer chemotherapy. Resistance of vaccinia virus towards the topoisomerase II (topo II) targeting anti-cancer drug etoposide has been mapped to the viral DNA ligase gene. The present study was performed to elucidate if the DNA ligase activity, besides topo II levels, was altered in human lymphatic leukaemia cell strains with different levels of etoposide resistance. At measurements of DNA ligase activity with specific substrates, to distinguish between different DNA ligases, a reduced DNA ligase activity was observed in the resistant substrains. In contrast, the initial step of the ligation process, formation of DNA ligase--AMP complex, did not decrease in the resistant cell strains, suggesting an alteration in a later reaction leading to a deteriorated DNA ligation. The results suggest that decreased DNA ligase activity, besides topo II alterations, may contribute to etoposide resistance of the investigated CEM cells. The relevance of this finding will be further investigated.