Son Nguyen

Human DNA ligases I and III, but not ligase IV, are required for microhomology-mediated end joining of DNA double-strand breaks

DNA nonhomologous end-joining (NHEJ) and homologous recombination are two distinct pathways of DNA double-strand break repair in mammalian cells. Biochemical and genetic studies showed that DNA ends can also be joined via microhomology-mediated end joining (MHEJ), especially when proteins responsible for NHEJ, such as Ku, are reduced or absent. While it has been known that Ku-dependent NHEJ requires DNA ligase IV, it is unclear which DNA ligase(s) is required for Ku-independent MHEJ. In this study, we used a cell-free assay to determine the roles of DNA ligases I, III and IV in MHEJ and NHEJ. We found that siRNA mediated down-regulation of DNA ligase I or ligase III in human HTD114 cells led to impaired end joining that was mediated by 2-, 3- or 10-bp microhomology. In addition, nuclear extract from human fibroblasts harboring a mutation in DNA ligase I displayed reduced MHEJ activity. Furthermore, treatment of HTD114 nuclear extracts with an antibody against DNA ligase I or III also significantly reduced MHEJ. These data indicate that DNA ligases I and III are required in MHEJ. DNA ligase IV, on the contrary, is not required in MHEJ but facilitates Ku-dependent NHEJ. Therefore, MHEJ and NHEJ require different DNA ligases.

Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells

The maintenance of genomic integrity is crucial to embryonic stem cells (ESC) considering the potential for propagating undesirable mutations to the resulting somatic and germ cell lineages. Indeed, mouse ESC (mESC) exhibit a significantly lower mutation frequency compared to differentiated cells. This could be due to more effective elimination of genetically damaged cells via apoptosis, or especially robust, sequence-conserving DNA damage repair mechanisms such as homologous recombination (HR). We used fluorescence microscopy and 3-dimensional image analysis to compare mESC and differentiated cells, with regard to HR-mediated repair of spontaneous and X-ray-induced double-strand breaks (DSBs). Microscopic analysis of repair foci, flow cytometry, and functional assays of the major DSB repair pathways indicate that HR is greater in mESC compared to fibroblasts. Strikingly, HR appears to be the predominant pathway choice to repair induced or spontaneous DNA damage throughout the ESC cycle in contrast to fibroblasts, where it is restricted to replicated chromatin. This suggests that alternative templates, such as homologous chromosomes, are more frequently used to repair DSB in ESC. Relatively frequent HR utilizing homolog chromosome sequences preserves genome integrity in ESC and has distinctive and important genetic consequences to subsequent somatic and germ cell lineages.

Reduced Apoptosis and Increased Deletion Mutations at Aprt Locus In vivo in Mice Exposed to Repeated Ionizing Radiation

Exposure to ionizing radiation (IR) is a risk factor for carcinogenesis because it is a mutagen. However, a single 4-Gy whole body X-ray exposure only induced a modest increase of mutations at the Aprt reporter gene locus in mouse T cells. Intriguingly, when the same dose of IR was given in a fractionated protocol (1 Gy x 4 at weekly intervals), there was a strong induction of Aprt mutations in T cells. Many of these were mutations that arose via interstitial deletions inclusive of Aprt or by intragenic deletions. We hypothesized that the weekly fractionated X-ray exposures select for somatic cells with reduced p53 expression and/or reduced apoptosis, which, in turn, may have facilitated the accumulation of interstitial deletions, as in p53-deficient mice. We indeed found that splenocytes of mice with three previous exposures (1 Gy x 4 in total) were more resistant to X-ray-induced apoptosis than those of mice exposed to X-rays for the first time (1 Gy total). Thus, repeated X-ray radiation selects for reduced apoptosis in vivo. However, this reduced apoptosis is p53-independent, because p53 induction and the up-regulation of genes downstream of p53, such as Bax and p21, were similar between the 1-Gy and 1 Gy x 4 groups. Reduced apoptosis probably allows the generation of more mutations, particularly deletion mutations. Because both reduced apoptosis and increased somatic mutation are risk factors for carcinogenesis, they may contribute to the paradigm in which different radiation exposure schemes are varied in their efficiency in inducing lymphomagenesis.

A human cell-based reporter detects microhomology-mediated end joining

DNA double-strand breaks (DSBs) are most often repaired by two pathways in mammalian cells, homologous recombination or non-homologous end joining. Biochemical and genetic studies showed that DSBs can also be joined via microhomology-mediated end joining (MHEJ), which is always mutagenic and may result in diseases, such as cancer. In this study we established a human cell-based reporter system to determine the prevalence of MHEJ events and factors that modulate MHEJ. A nonfunctional puromycin acetyltransferase (Pac) gene, disrupted by an insertion flanked by two microhomologous repeats, was integrated into chromosomes of human HT1080 cells. Repair of DSBs via MHEJ using the repeats resulted in deletion of the insertion and restoration of the Pac gene function, thus rendering the cells puromycin resistant. Our results showed that MHEJ spontaneously occurs at the reporter locus (loci), manifested by formation of puromycin resistant (puro(r)) colonies after culturing reporter cells in medium containing puromycin. The frequency of puro(r) cells can be greatly increased by site-directed DSB inside the insertion. Our results also demonstrated that the frequency of puro(r) cells is affected by the length of the repeat and by the size of the intervening sequence. Thus, this cell-based assay provides a platform for evaluating factors modulating in vivo MHEJ.