Biomedical subjects
Publications and source records attributed to J E Cleaver.
The distribution of labeled chromatids at the second mitosis after labeling with tritiated thymidine is random in both Vicia faba (the broad bean) and Potorous tridactylis (the rat kangaroo). This finding is contrary to that predicted by the hypothesis that chromatids containing "grandparent" polynucleotide templates segregate from those containing "parent" templates.
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Poly(ADP-ribose) is a nuclear polymer that is synthesized in response to DNA-strand breaks and covalently modifies numerous nuclear proteins. Inhibition of poly(ADP-ribose) polymerase by 3-amino-benzamide in cells exposed to DNA-damaging agents has a variety of cellular effects, including increases in cell killing, frequency of single-strand breaks, repair replication, and sister-chromatid exchange. These increases have been interpreted as an indication that poly(ADP-ribose) polymerization regulates the rate of ligation. Because of slow ligation, continued repair polymerization should therefore generate longer repair patches. Direct measurement of the rate of ligation of intracellular repair patches and of the size of repair patches indicates that they are unchanged when poly(ADP-ribose) polymerization is inhibited. We therefore conclude that poly(ADP-ribose) does not regulate the ligation stage of repair but instead may regulate the activity of intracellular nucleases and other enzymes that can cause additional DNA damage and changes in chromatin structure.
We have created a cell line that can repair damage in chromosomal DNA and in herpes virus, while not repairing the same damage in shuttle vectors (pZ189 and pRSVcat). This cell line, a xeroderma pigmentosum (XP) revertant, repairs the minor (6-4)-photoproducts, but not cyclobutane dimers, in chromosomal DNA. The phenotype of this revertant after irradiation with ultraviolet (UV) light is the same as that of normal cells for survival, repair replication, recovery of rates of DNA and RNA synthesis, and sister-chromatid exchange formation, which indicates that a failure to mend cyclobutane dimers may be irrelevant to the fate of irradiated human cells. The two shuttle vectors were grown in Escherichia coli and assayed during transient passage in human cells, whereas the herpes virus was grown and assayed exclusively in mammalian cells. The ability of the XP revertant to distinguish between the shuttle vector and herpes virus DNA molecules according to their 'cultural background', i.e., bacterial or mammalian, may indicate that one component of the repair of UV damage involves gene products that recognize DNA markers that are uniquely mammalian, such as DNA methylation patterns. This component of excision repair may be involved in the original defect and the reversion of XP group A cells.
3-Aminobenzamide is commonly assumed to be a specific inhibitor of poly(ADP-ribose) polymerase. However, it also inhibits de novo synthesis of DNA purines at concentrations of 5 mM and above. A mutant cell line that lacks the ability to incorporate exogenous purines is therefore exceptionally sensitive to the toxic effects of 3-aminobenzamide. This finding has implications for the underlying mechanism of sensitivity to 3-aminobenzamide recently reported in a cell line from an immunodeficient patient, and also implies that many studies using high concentrations of 3-aminobenzamide may not demonstrate a role for poly(ADP-ribose) because of artifacts arising from disturbance of intracellular purine metabolism.
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We describe here a model for sequential recruitment of various enzymatic systems that maintain DNA replication fidelity in cells with damaged bases, especially those formed by ultraviolet (UV) irradiation. Systems of increasing complexity but decreasing fidelity are recruited to restore replication of damaged DNA. The first and most accurate response is nucleotide excision repair (NER) that is cell cycle-independent; next come various delaying cell cycle checkpoints that provide an extended time window for NER. These delay the onset of the S phase at the G1/S boundary, and inhibit the initiation of individual replicating units (replicons and clusters of replicons) within the S phase. When checkpoints fail to operate completely, DNA replication forks must negotiate damage and the loss of coding information on the parental DNA strands. Replication can be resumed using bypass polymerases, or alternative bypass mechanisms. Finally, if all else fails, replication forks may degrade to double strand breaks and recombinational processes then allow their reconstruction. A network of signaling kinases modulates the efficiency of many damage responsive proteins to tailor their activities and subcellular localizations by phosphorylation and dephosphorylation.