Serum effects on DNA repair in human cells

Freshly isolated human lymphocytes were used to determine how serum supplements affect cellular capacity to repair UV damage. Repair capacity was always found to be greatest in medium supplemented with autologous plasma. Variability in repair capacity among individuals was greater in serum supplemented medium than in unsupplemented medium. Thus, in vitro cellular responses will most accurately represent in vivo responses if autologous serum factors are present in the culture medium. This is of particular importance in studies attempting to correlate DNA repair capacity with age or susceptibility to carcinogenesis.     

Acetylation regulates DNA repair mechanisms in human cells

The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation.     

Heteroduplex repair in extracts of human HeLa cells

A general repair process for DNA heteroduplexes has been detected in HeLa cell extracts. Using a variety of M13mp2 DNA substrates containing single-base mismatches and extra nucleotides, extensive repair is observed after incubation with HeLa cell cytoplasmic extracts and subsequent transfection of bacterial cells with the treated DNA. Most, but not all, mispairs as well as two frameshift heteroduplexes are repaired efficiently. Parallel measurements of repair in HeLa extracts and in Escherichia coli suggest that repair specificities are similar for the two systems. The presence of a nick in the molecule is required for efficient repair in HeLa cell extracts, and the strand containing the nick is the predominantly repaired strand. Mismatch-dependent DNA synthesis is observed when radiolabeled restriction fragments, produced by reaction of the extract with heteroduplex and homoduplex molecules, are compared. Specific labeling of fragments, representing a region of approximately 1,000 base pairs and containing the nick and the mismatch, is detected for the heteroduplex substrate but not the homoduplex. The repair reaction is complete after 20 min and requires added Mg2+, ATP, and an ATP-regenerating system, but not dNTPs, which are present at sufficient levels in the extract. An inhibitor of DNA polymerase beta, dideoxythimidine 5'-triphosphate, does not inhibit mismatch-specific DNA synthesis. Aphidicolin, an inhibitor of DNA polymerases alpha, delta, and epsilom, inhibits both semiconservative replication and repair synthesis in the extract. Butylphenyl-dGTP also inhibits both replicative and repair synthesis but at a concentration known to inhibit DNA polymerase alpha preferentially rather than delta or epsilon. This suggests that DNA polymerase alpha may function in mismatch repair.     

Interaction of nick-directed DNA mismatch repair and loop repair in human cells

In human cells, large DNA loop heterologies are repaired through a nick-directed pathway independent of mismatch repair. However, a 3'-nick generated by bacteriophage fd gene II protein heterology is not capable of stimulating loop repair. To evaluate the possibility that a mismatch near a loop could induce both repair types in human cell extracts, we constructed and tested a set of DNA heteroduplexes, each of which contains a combination of mismatches and loops. We have demonstrated that a strand break generated by restriction endonucleases 3' to a large loop is capable of provoking and directing loop repair. The repair of 3'-heteroduplexes in human cell extracts is very similar to that of 5'-heteroduplex repair, being strand-specific and highly biased to the nicked strand. This observation suggests that the loop repair pathway possesses bidirectional repair capability similar to that of the bacterial loop repair system. We also found that a nick 5' to a coincident mismatch and loop can apparently stimulate the repair of both. In contrast, 3'-nick-directed repair of a G-G mismatch was reduced when in the vicinity of a loop (33 or 46 bp between two sites). Increasing the distance separating the G-G mismatch and loop by 325 bp restored the efficiency of repair to the level of a single base-base mismatch. This observation suggests interference between 3'-nick-directed large loop repair and conventional mismatch repair systems when a mispair is near a loop. We propose a model in which DNA repair systems avoid simultaneous repair at adjacent sites to avoid the creation of double-stranded DNA breaks.     

DNA repair in human pluripotent stem cells is distinct from that in non-pluripotent human cells

The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use.     

Cell cycle regulation of DNA repair in normal and repair deficient human cells

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylmethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14–15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2–4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.     

AP endonuclease-independent DNA base excision repair in human cells

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.     

Spontaneous DNA repair in human mononuclear cells is calcium-dependent

Spontaneous DNA repair in peripheral blood mononuclear cells (PBMC) has been recently described. The aim of this study was to evaluate whether spontaneous DNA repair is Ca(2+)-dependent, as in vitro-stimulated DNA repair. Spontaneous DNA repair in PBMC was measured in a 1mM Ca2+ medium. The effect of extracellular Ca2+ chelation by EGTA, intracellular Ca2+ chelation by bapta-AM, and Ca2+ loading by the ionophore A23187 was examined. The signal transduction pathway was evaluated by inhibiting protein tyrosine kinase with genistein, calmodulin with W7, and calcineurin with cyclosporin A and tacrolimus. Extracellular Ca2+ chelation had no effect on spontaneous DNA repair, while both intracellular chelation and calcium overloading inhibited the DNA repair. Inhibition of protein tyrosine kinase, calmodulin or calcineurin reduced DNA repair. In conclusion, spontaneous DNA repair is mainly Ca(2+)-dependent at a narrow range of intracellular Ca2+ concentrations. The signal transduction cascade includes protein tyrosine kinase, calmodulin, and calcineurin.     

Spontaneous DNA repair in human peripheral blood mononuclear cells

DNA molecules are constantly damaged during mitosis and by oxygen-free radicals produced by either cellular metabolism or by external factors. Populations at risk include patients with cancer-prone disease, patients under enhanced oxidative stress, and those treated with immunosuppressive/cytotoxic therapy. The DNA repair process is crucial in maintaining the genomal DNA integrity. The aim of this study was to evaluate spontaneous DNA repair capacity of peripheral blood mononuclear cells (PBMC) from normal blood donors. PBMC DNA repair ability represents DNA repair by other tissues as well. It is shown in the present study that in vitro incorporation of [3H]thymidine in non-stimulated PBMC expresses the ability of the cells to repair DNA damage. This method was validated by double-stranded DNA measurements. Both catalase and Fe2+ increased DNA repair, the former by preventing re-breakage of newly repaired DNA and the latter by introducing additional DNA damage, which enhanced DNA repair. Better understanding of DNA repair processes will enable to minimize DNA damage induced by oxidative stress.     

Mechanisms of impairment of DNA repair in human cells. Interferons stimulated DNA repair in xeroderma pigmentosum cells

DNA repair synthesis and strand break DNA repair induced by 4-nitroquinoline-1-oxide and UV-irradiation in Xeroderma pigmentosum lymphocytes and fibroblasts pretreated by leucocyte interferons were studied. Stimulation of DNA repair synthesis in interferon-pretreated Xeroderma pigmentosum cells, defective in incision, was detected. No such effect was noted for strand break DNA repair. Hence, antimutagenic activity of interferons in human cells is connected with their modificating effect on DNA repair.