Levels Of DNA Ligase Research Articles

Functional analysis of XRCC4 mutations in reported microcephaly and growth defect patients in terms of radiosensitivity.

Non-homologous end joining is one of the main pathways for DNA double-strand break (DSB) repair and is also implicated in V(D)J recombination in immune system. Therefore, mutations in non-homologous end-joining (NHEJ) proteins were found to be associated with immunodeficiency in human as well as in model animals. Several human patients with mutations in XRCC4 were reported to exhibit microcephaly and growth defects, but unexpectedly showed normal immune function. Here, to evaluate the functionality of these disease-associated mutations of XRCC4 in terms of radiosensitivity, we generated stable transfectants expressing these mutants in XRCC4-deficient murine M10 cells and measured their radiosensitivity by colony formation assay. V83_S105del, R225X and D254Mfs*68 were expressed at a similar level to wild-type XRCC4, while W43R, R161Q and R275X were expressed at even higher level than wild-type XRCC4. The expression levels of DNA ligase IV in the transfectants with these mutants were comparable to that in the wild-type XRCC4 transfectant. The V83S_S105del transfectant and, to a lesser extent, D254Mfs*68 transfectant, showed substantially increased radiosensitivity compared to the wild-type XRCC4 transfectant. The W43R, R161Q, R225X and R275X transfectants showed a slight but statistically significant increase in radiosensitivity compared to the wild-type XRCC4 transfectant. When expressed as fusion proteins with Green fluorescent protein (GFP), R225X, R275X and D254Mfs*68 localized to the cytoplasm, whereas other mutants localized to the nucleus. These results collectively indicated that the defects of XRCC4 in patients might be mainly due to insufficiency in protein quantity and impaired functionality, underscoring the importance of XRCC4’s DSB repair function in normal development.

Abstract 5495: ALT NHEJ is a therapeutic target in hormone therapy resistant ER/PR/HER+ and ER/PR/HER- breast cancers

Abstract Approximately 75% of breast cancers are estrogen and/or progesterone positive (ER/PR+) and can be treated in the clinic with anti-hormone therapy; however, resistance is an increasing problem. ER/PR negative (−) breast cancers, on the other hand, have few treatment options. Therefore, novel therapies are required for therapy resistant ER/PR+ and ER/PR- breast cancers. Breast cancers have abnormal responses to the repair of DNA double strand breaks (DSBs) leading to the generation of structural chromosomal abnormalities and genomic instability. DSBs are repaired by at least 2 pathways: homologous recombination (HR) that performs error-free repair and non-homologous end-joining (NHEJ) that is error-prone. An alternative (ALT) NHEJ pathway has been identified that is associated with the generation of chromosomal translocations, deletions and genomic instability. We have recently shown that with respect to non-tumorigenic MCF10A, ER/PR+ MCF7 breast cancer cells express reduced levels of DNA ligase IV, a component of the classic (C) NHEJ pathway, whereas, expression of DNA ligase IIIα, a component of ALT NHEJ, is increased. To determine whether abnormal ALT NHEJ may be a target for therapy in hormone resistant ER/PR+ and ER/PR- patients, we examined steady state levels of C- and ALT-NHEJ proteins in tamoxifen (TAM 1,2,3) and aromatase (LTLT) resistant cell lines derived from MCF7 cells and ER/PR- cells (MDAMB231, SKBR3). Only ER/PR- cells show further reductions in DNA ligase IV and Ku70 compared with MCF10A. Both hormone resistant ER/PR+ and ER/PR- cells show further increases in ALT NHEJ proteins DNA ligase IIIα, poly- (ADP-ribose) polymerase (PARP1) and ALT NHEJ activity as measured by established NHEJ assays compared to controls. Since PARP1 inhibitors have been shown to specifically kill cells with defects in DSB repair and sporadic breast cancer cells have upregulated PARP1 and DNA ligase IIIα, we examined the effect of PARP1 inhibitor ABT888 in ER/PR+ and ER/PR- cells in the presence or absence of a DNA ligase I/III inhibitor. While either inhibitor alone had little effect on cell survival, both therapy resistant ER/PR+ and ER/PR- cells are highly sensitive to a combination of DNA repair inhibitors, acting synergistically. This treatment also decreases ALT NHEJ repair, demonstrating specific targeting of this pathway. Importantly, MCF10A is insensitive to these inhibitors. Finally, siRNA knockdown of C NHEJ protein Ku70 in hormone therapy sensitive ER/PR+ MCF7 increases ALT NHEJ repair activity and sensitivity to the combination of DNA repair inhibitors. Intriguingly, MCF7 cells with increased ALT NHEJ activity also acquire resistance to hormone therapy. Thus, ALT NHEJ appears to be linked to resistance to hormone therapy and is a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5495. doi:10.1158/1538-7445.AM2011-5495

Concomitant reversion of the characteristic phenotypic properties of a cell line of Bloom's syndrome origin.

The fibroblast strain GM3498 and the lymphoblastoid line GM4408 have been established from the same Bloom's syndrome patient, as confirmed by DNA hybridization with hypervariable probes and isoenzyme analysis. Strain GM3498, but not GM4408, exhibits a high frequency of spontaneous sister chromatid exchange, hypersensitivity to ethyl methanesulphonate and an anomalously low level of DNA ligase I activity. We conclude that GM3498 is representative of Bloom's syndrome, whereas GM4408 is a revertant cell line. These data agree with a model in which a malfunctioning DNA ligase I is the primary cause of the syndrome.

Periodic transcription as a means of regulating gene expression during the cell cycle: contrasting modes of expression of DNA ligase genes in budding and fission yeast.

Using cultures synchronised by three independent procedures, we have shown that the CDC9 gene, coding for DNA ligase, is periodically expressed in the Saccharomyces cerevisiae cell cycle. The level of CDC9 transcript increases many fold in late G1 reaching a peak at about the G1/S phase boundary and preceding the peak in histone message by some 20 min. The level of DNA ligase itself also fluctuates, showing the expected pattern for a stable enzyme synthesised periodically. In contrast, the transcript from the DNA ligase gene (CDC17) of Schizosaccharomyces pombe is present at a constant level throughout the cell cycle, and no fluctuation in amount was detected, although the histone H2A showed the expected periodic synthesis. Furthermore, DNA ligase activity remains at a constant level during the S. pombe cell cycle showing that there is unlikely to be any form of translational control. These contrasting modes of expression of the DNA ligase genes in the two organisms suggests that when periodic transcription is observed from an essential cell cycle gene, it may have no particular significance for regulating progress through the cell cycle. Also, regulatory circuits may be less well conserved between organisms than the processes they control and thus different organisms may utilise quite different modes of control to achieve the same ends.