ALTERED DNA LIGASE I IN CANCER-PRONE HEREDITARY DISEASE

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Cells from patients with Bloom's syndrome (BS), an autosomal inherited disorder associated with increased cancer frequency, exhibit chromosomal abnormalities. These include an increased rate of spontaneous sister chromatid exchanges (SCE) and chromosomal aberrations; together with slow replicon fork progression and a retarded rate of DNA chain maturation; and slightly increased sensitivity to DNA damaging agents. Several of these features are also characteristic of E. coli and yeast mutants with a defective DNA ligase. We recently found that DNA ligase I activity was defective in BS cells, whereas the activities of DNA ligase II, DNA polymerase- alpha and -beta were not altered. The inability to properly ligate DNA breaks during replication, repair, or recombination may account for the many disorders of BS, including the greatly increased risk of cancer. The aim of this proposal is: 1) to determine if the decrease in enzyme activity correlates with the decrease in protein molecules in BS cells using immunoblot, ligase-AM(32P) adduct assay, and SDS-gel electrophoresis; 2) to purify the enzymes, compare the structural and biochemical properties of normal and aberrant ligase I, raise antibodies, and to identify the altered domain; 3) to screen other cancer-prone cells, mutants, normal-BS cell hybrids, and pre-neoplastic tissues for abnormalities in ligases; and finally, 4) to clone the normal and aberrant ligase I genes, and correct the BS defect by microinjection of ligase protein and/or transfection of the ligase gene. The elucidation of the molecular nature of cancer-prone genetic diseases may well provide new insights into the mechanism of DNA processing and deduction of the obligatory step(s) in the initiation of human cancer.