A Stochastic Model of DNA Fragments Rejoining

When cells are exposed to ionizing radiation, DNA damages in the form of single strand breaks (SSBs), double strand breaks (DSBs), base damage or their combinations are frequent events. It is known that the complexity and severity of DNA damage depends on the quality of radiation, and the microscopic dose deposited in small segments of DNA, which is often related to the linear transfer energy (LET) of the radiation. Experimental studies have suggested that under the same dose, high LET radiation induces more small DNA fragments than low-LET radiation, which affects Ku efficiently binding with DNA end and might be a main reason for high-LET radiation induced RBE [1] since DNA DSB is a major cause for radiation-induced cell death. In this work, we proposed a mathematical model of DNA fragments rejoining according to non-homologous end joining (NHEJ) mechanism. By conducting Gillespie''s stochastic simulation, we found several factors that impact the efficiency of DNA fragments rejoining. Our results demonstrated that aberrant DNA damage repair can result predominantly from the occurrence of a spatial distribution of DSBs leading to short DNA fragments. Because of the low efficiency that short DNA fragments recruit repair protein and release the protein residue after fragments rejoining, Ku-dependent NHEJ is significantly interfered with short fragments. Overall, our work suggests that inhibiting the Ku-dependent NHEJ may significantly contribute to the increased efficiency for cell death and mutation observed for high LET radiation.     

Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 (SMCHD1) Promotes Non-homologous End Joining and Inhibits Homologous Recombination Repair upon DNA Damage

Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) has been shown to be involved in gene silencing and DNA damage. However, the exact mechanisms of how SMCHD1 participates in DNA damage remains largely unknown. Here we present evidence that SMCHD1 recruitment to DNA damage foci is regulated by 53BP1. Knocking out SMCHD1 led to aberrant γH2AX foci accumulation and compromised cell survival upon DNA damage, demonstrating the critical role of SMCHD1 in DNA damage repair. Following DNA damage induction, SMCHD1 depletion resulted in reduced 53BP1 foci and increased BRCA1 foci, as well as less efficient non-homologous end joining (NHEJ) and elevated levels of homologous recombination (HR). Taken together, these results suggest an important function of SMCHD1 in promoting NHEJ and repressing HR repair in response to DNA damage.     

耐辐射球菌DNA双链断裂修复机制研究新进展

耐辐射球菌对于电离辐射等DNA损伤剂具有极强的抗性,能够将同一个基因组中同时产生的高达100个以上的DNA双链断裂在数十小时内高效而精准地进行修复,是研究DNA双链断裂修复机制的重要模式生物。同源重组、非同源末端连接和单链退火途径作为3个主要的修复途径参与了耐辐射球菌基因组DNA双链断裂的修复过程。此外,一系列新发现的重要蛋白质,如Ppr I、Ddr B等对于耐辐射球菌基因组的修复过程同样至关重要。根据本实验室和国内外在这一研究领域近年来的报道,以不同的修复途径为线索,综述该菌DNA双链断裂修复机制的最新研究成果。     

Absence of Ku70 gene obliterates X-ray-induced lacZ mutagenesis of small deletions in mouse tissues

With the goal of understanding the role of non-homologous end-joining repair in the maintenance of genetic information at the tissue level, we studied mutations induced by radiation and subsequent repair of DNA double-strand breaks in Ku70 gene-deficient lacZ transgenic mice. The local mutation frequencies and types of mutations were analyzed on a lacZ gene that had been chromosomally integrated, which allowed us to monitor DNA sequence alterations within this 3.1-kbp region. The mutagenic process leading to the development of the most frequently observed small deletions in wild-type mice after exposure to 20 Gy of X rays was suppressed in Ku70(-/-) mice in the three tissues examined: spleen, liver and brain. Examination of DNA break rejoining and the phosphorylation of histone H2AX in Ku70-deficient and -proficient mice revealed that Ku70 deficiency decreased the frequency of DNA rejoining, suggesting that DNA rejoining is one of the causes of radiation-induced deletion mutations. Limited but statistically significant DNA rejoining was found in the liver and brain of Ku70-deficient mice 3.5 days after irradiation, showing the presence of a DNA double-strand break repair system other than non-homologous end joining. These data indicate a predominant role of non-homologous end joining in the production of radiation-induced mutations in vivo.     

The Ku-dependent non-homologous end-joining but not other repair pathway is inhibited by high linear energy transfer ionizing radiation

Ionizing radiation (IR) induced DNA double strand breaks (DSBs) are repaired by both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR) in mammalian cells. The NHEJ repair includes a Ku-dependent main pathway and a PARP-1-dependent complementary pathway. Compared with low linear energy transfer (LET) IR (X or gamma ray) at the same doses, high LET IR (high-charge particles) induces more cell death because of ineffective DNA repair. However, it remains unclear whether high LET IR inhibits all repair or specifically one repair pathway. By combining the assays of clonogenic survival, G2M checkpoint and gammaH2AX in the cell lines with deficiencies in different repair genes, we show here that high LET IR inhibits only the Ku-dependent main NHEJ pathway and does not inhibit either the HRR pathway or the PARP-1-dependent complementary NHEJ pathway. In addition, by developing an assay to detect small fragments of DSB (<400 bp) and by detecting the binding abilities of purified Ku and PARP to different sized dsDNA, we present a possible link for explaining the phenotypes. When compared with low LET IR at the same dose, high LET IR might induce similar yields of DNA DSBs in total but it might induce more small fragments of DNA DSBs (<40 base pairs) that prevent Ku binding efficiently to two ends of one DSB fragment at the same time, thus delaying Ku-dependent repair.     

组蛋白去乙酰化酶抑制剂对DNA双链断裂修复路径的作用

DNA双链断裂(double strand breaks, DSBs)对细胞生存是致命的.细胞内非同源末端连接(NHEJ)、重组修复(HDR)、单链退火修复(SSA)和微同源序列末端连接(MMEJ)等通路可竞争性修复DNA双链断裂损伤.在肿瘤细胞DNA中制造难以修复的基因损伤,诱导肿瘤细胞周期中止、坏死和凋亡是临床放、化疗的主要策略.组蛋白去乙酰化酶（histone deacetylase）作为抗肿瘤治疗的新靶标,其抑制剂(histonedeacetylase inhibitors, HDACi)可显著降低肿瘤细胞DSBs修复能力,增强肿瘤细胞的放、化疗敏感性.研究显示,HDACi抑制了肿瘤细胞中具有正确修复倾向的HDR和经典NHEJ通路,具有错误修复倾向的SSA和MMEJ路径也可能牵涉其中.目前,HDACi作用于DSBs修复通路的分子机制已取得较大进展,但仍有许多问题有待阐明.     

PP6 regulatory subunit R1 is bidentate anchor for targeting protein phosphatase-6 to DNA-dependent protein kinase

DNA-dependent protein kinase (DNA-PK) becomes activated in response to DNA double strand breaks, initiating repair by the non-homologous end joining pathway. DNA·PK complexes with the regulatory subunit SAPSR1 (R1) of protein phosphatase-6 (PP6). Knockdown of either R1 or PP6c prevents DNA-PK activation in response to ionizing radiation-induced DNA damage and radiosensitizes glioblastoma cells. Here, we demonstrate that R1 is necessary for and bridges the interaction between DNA-PK and PP6c. Using R1 deletion mutants, DNA-PK binding was mapped to two distinct regions of R1 spanning residues 1-326 and 522-700. Either region expressed alone was sufficient to bind DNA-PK, but only deletion of residues 1-326, not 522-700, eliminated interaction of R1 with DNA-PK. We assign 1-326 as the dominant domain and 522-700 as the supporting region. These results demonstrate that R1 acts as a bidentate anchor to DNA-PK and recruits PP6c. Targeting the dominant interface with small molecule or peptidomimetic inhibitors could specifically prevent activation of DNA-PK and thereby sensitize cells to ionizing radiation and other genotoxic agents.     

Ionizing radiation sensitivity of DNA polymerase lambda-deficient cells

Ionizing radiation induces a diverse spectrum of DNA lesions, including strand breaks and oxidized bases. In mammalian cells, ionizing radiation-induced lesions are targets of non-homologous end joining, homologous recombination, and base excision repair. In vitro assays show a potential involvement of DNA polymerase lambda in non-homologous end joining and base excision repair. In this study, we investigated whether DNA polymerase lambda played a significant role in determining ionizing radiation sensitivity. Despite increased sensitivity to hydrogen peroxide, lambda-deficient mouse embryonic fibroblasts displayed equal survival after exposure to ionizing radiation compared to their wild-type counterparts. In addition, we found increased sensitivity to the topoisomerase inhibitors camptothecin and etoposide in the absence of polymerase lambda. These results do not reveal a major role for DNA polymerase lambda in determining radiosensitivity in vivo.     

Induction of homologous recombination in the hprt gene of V79 Chinese hamster cells in response to low- and high-LET irradiation

Dense ionization tracks from high linear energy transfer (LET) radiations form multiple damaged sites (MDS), which involve several types of DNA lesions in close vicinity. The primary DNA damage triggers sensor proteins that activate repair processes, cell cycle control or eventually apoptosis in subsequent cellular responses. The question how homologous recombination (HR) and non-homologous end joining (NHEJ) interact in the repair of radiation-induced DNA damage of MDS type has been addressed in different model systems but several questions remain to be answered. We have therefore challenged cells with treatments of ionizing radiation of different qualities to investigate whether primary DNA damages of different complexity are reflected in the processes of repair by HR as well as cell survival. We used the V79 derived SPD8 cell line to determine the induction of HR in the hprt exon 7 and clonogenic assay for survival in response to radiation. SPD8 cells were irradiated with gamma-rays (137Cs 0.5 keV/microm), boron ions (40 and 80 keV/microm) and nitrogen ions (140 keV/microm), with doses up to 5 Gy. Analysis of clonogenic survival showed that B-ions (80 keV/microm) and N-ions were more toxic than gamma-rays, 4.1 and 5.0 times respectively, while B-ions at 40 keV/microm were 2.0 times as toxic as gamma-rays. Homologous recombination in the cells exposed to B-ions (80 keV/microm) increased 2.9 times, a significant response as compared to cells exposed to gamma-rays, while for B-ions (40 keV/microm) and N-ions a nonsignificant increase in HR of 1.2 and 1.4, respectively, was observed. We hypothesize that the high-LET generated formation of MDS is responsible for the enhanced cytotoxicity as well as for the mobilization of the HR machinery.     

Rapid assessment of two major repair activities against DNA double-strand breaks in vertebrate cells

A linearized plasmid DNA, in which tandem repeats of 400bp flank the breakpoints, was transfected into vertebrate cells, and breakpoint junctions of plasmid DNA circularized in the cells were analyzed to assess the repair activities against DNA double-strand break (DSB) by non-homologous end joining and homology-directed repair (i.e., homologous recombinational repair and single-strand annealing). The circularization by non-homologous end joining repair of the breakpoints depended on the expression of DNA-PKcs, while that by homology-directed repair through the repeats depended on the length of the repeats, indicating that these two DSB repair activities can be rapidly assessed by this assay. Predominance in circularization by either non-homologous end joining or homology-directed repair differed among cells examined, and circularization was exclusively undertaken by homology-directed repair in DT40 cells known to show a high homologous recombination rate against gene-targeting vectors. Thus, this assay will be helpful in studies on mechanisms and inter-cellular variations of DSB repair.     

原核生物的NHEJ修复途径

DNA双链断裂(DSBs)是严重的DNA损伤形式之一,生物体对DSBs的修复可通过同源重组(HR)或非同源末端连接途径(NHEJ)进行。长期以来,人们普遍认为HR是细菌DSBs修复的惟一途径,但在分支杆菌和其它原核生物体内NHEJ途径的发现,使这一观念得以颠覆。最近的研究表明,细菌NHEJ修复系统是一个双组分系统,包含一个多功能的DNA连接酶(LigD)和DNA末端结合蛋白Ku,具有DSBs修复所需的断裂末段识别、末端加工和连接活性。重点综述细菌NHEJ修复系统的组成、结构以及生理功能。     

Sirtuin家族与DNA损伤修复

DNA损伤的发生与积累是造成细胞功能紊乱的根本原因,也是引起衰老与肿瘤等疾病发生的关键事件。为维持机体自身遗传物质的完整性与稳定性,生物体内拥有多种针对不同类型DNA损伤的修复方式。Sirtuin蛋白是一组NAD+依赖的、高度保守的组蛋白去乙酰化酶,可通过去乙酰化作用调节众多底物蛋白质的表达、活性与稳定性。 近来的研究显示,DNA损伤修复途径的多个关键蛋白质是Sirtuin的下游底物。Sirtuin蛋白通过调节同源重组修复、非同源末端修复、核苷酸切除修复等途径中的核心蛋白质参与修复包括双链断裂（double stranded breakes, DSBs）在内的多种DNA损伤类型,从而在维持基因组稳定性、寿命以及细胞能量代谢调节等一系列生物学作用中发挥至关重要的作用。本综述将介绍近年来Sirtuin与DNA损伤修复的研究进展。     

Cell cycle delay in murine pre-osteoblasts is more pronounced after exposure to high-LET compared to low-LET radiation

Space radiation contains a complex mixture of particles comprised primarily of protons and high-energy heavy ions. Radiation risk is considered one of the major health risks for astronauts who embark on both orbital and interplanetary space missions. Ionizing radiation dose-dependently kills cells, damages genetic material, and disturbs cell differentiation and function. The immediate response to ionizing radiation-induced DNA damage is stimulation of DNA repair machinery and activation of cell cycle regulatory checkpoints. To date, little is known about cell cycle regulation after exposure to space-relevant radiation, especially regarding bone-forming osteoblasts. Here, we assessed cell cycle regulation in the osteoblastic cell line OCT-1 after exposure to various types of space-relevant radiation. The relative biological effectiveness (RBE) of ionizing radiation was investigated regarding the biological endpoint of cellular survival ability. Cell cycle progression was examined following radiation exposure resulting in different RBE values calculated for a cellular survival level of 1 %. Our findings indicate that radiation with a linear energy transfer (LET) of 150 keV/μm was most effective in inducing reproductive cell killing by causing cell cycle arrest. Expression analyses indicated that cells exposed to ionizing radiation exhibited significantly up-regulated p21(CDKN1A) gene expression. In conclusion, our findings suggest that cell cycle regulation is more sensitive to high-LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression.     

Repair of ionizing radiation damage in mammalian cells. Alternative pathways and their fidelity

Ionizing radiation causes a variety of types of damage to DNA in cells, requiring the concerted action of a number of DNA repair enzymes to restore genomic integrity. The DNA base-excision repair and DNA double-strand break repair pathways are particularly important. While single base damages are rapidly excised and repaired using the opposite (undamaged) strand as a template, the correct repair of DNA double-strand breaks may present more difficulties to cellular enzymes owing to the loss of template. In the last few years evidence in support of several enzymatic pathways for the repair of such double-stranded damage has been found. At present we may distinguish at least three pathways: homologous recombination repair, non-homologous (DNA-PK-dependent) end joining, and repeat-driven end joining. This paper focuses on evidence for the first and third of these pathways, and considers in particular their relative importance in mammalian cells and implications for the fidelity of repair.     

Repair of Oxidative DNA Damage in Saccharomyces cerevisiae

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae.     

In vitro non-homologous DNA end joining assays—The 20th anniversary

DNA double-strand breaks (DSBs) are the most serious forms of DNA damage in cells. Unrepaired or misrepaired DSBs account for some of the genetic instabilities that lead to mutations or cell death, and consequently, to cancer predisposition. In human cells non-homologous DNA end joining (NHEJ) is the main repair mechanism of these breaks. Systems for DNA end joining study have been developing during the last 20 years. New assays have some advantages over earlier in vitro DSBs repair assays because they are less time-consuming, allow the use of clinical material and examination of the joining DNA ends produced physiologically in mammalian cells. Proteins involved in NHEJ repair pathway can serve as biomarkers or molecular targets for anticancer drugs. Results of studies on NHEJ in cancer could help to select potent repair inhibitors that may selectively sensitize tumor cells to ionizing radiation (IR) and chemotherapy. Here, we review the principles and practice of in vitro NHEJ assays and provide some insights into the future prospects of this assay in cancer diagnosis and treatment.     

The role of Schizosaccharomyces pombe Rad32, the Mre11 homologue, and other DNA damage response proteins in non-homologous end joining and telomere length maintenance.

The Schizosaccharomyces pombe homologue of Mre11, Rad32, is required for repair of UV- and ionising radiation-induced DNA damage and meiotic recombination. In this study we have investigated the role of Rad32 and other DNA damage response proteins in non-homologous end joining (NHEJ) and telomere length maintenance in S.pombe. We show that NHEJ in S.pombe occurs by an error-prone mechanism, in contrast to the accurate repair observed in Saccharomyces cerevisiae. Deletion of the rad32 gene results in a modest reduction in NHEJ activity and the remaining repair events that occur are accurate. Mutations in two of the phosphoesterase motifs in Rad32 have no effect on the efficiency or accuracy of end joining, suggesting that the role of Rad32 protein may be to recruit another nuclease(s) for processing during the end joining reaction. We also analysed NHEJ in other DNA damage response mutants and showed that the checkpoint mutant rad3-d and two recombination mutants defective in rhp51 and rhp54 (homologues of S.cerevisiae RAD51 and RAD54, respectively) are not affected. However disruption of rad22, rqh1 and rhp9 / crb2 (homologues of the S.cerevisiae RAD52, SGS1 and RAD9 genes) resulted in increased NHEJ activity. Telomere lengths in the rad32, rhp9 and rqh1 null alleles were reduced to varying extents intermediate between the lengths observed in wild-type and rad3 null cells.     

The Ku‐dependent non‐homologous end‐joining pathway contributes to low‐dose radiation‐stimulated cell survival

Low‐dose (≤0.1 Gy) radiation‐induced adaptive responses could protect cells from high‐challenge dose radiation‐induced killing. The protective role is believed to promote the repair of DNA double‐strand breaks (DSBs) that are a severe threat to cell survival. However, it remains unclear which repair pathway, homologous recombination repair (HRR) or non‐homologous end‐joining (NHEJ), is promoted by low‐dose radiation. To address this question, we examined the effects of low‐dose (0.1 Gy) on high‐challenge dose (2–4 Gy) induced killing in NHEJ‐ or HRR‐deficient cell lines. We showed that 0.1 Gy reduced the high‐dose radiation‐induced killing for wild‐type or HRR‐deficient cells, but enhanced the killing for NHEJ‐deficient cells. Interestingly, low‐dose radiation also enhanced the killing for wild‐type cells exposed to high‐challenge dose radiation with high‐linear energy transfer (LET). Because it is known that high‐LET radiation induces an inefficient NHEJ, these results support that the low‐dose radiation‐stimulated protective role in reducing high‐challenge dose (low‐LET)‐induced cell killing might depend on NHEJ. In addition, we showed that low‐dose radiation activated the DNA‐PK catalytic subunit (DNA‐PKcs) and the inhibitor of DNA‐PKcs destroyed the low‐dose radiation‐induced protective role. These results suggest that low‐dose radiation might promote NHEJ through the stimulation of DNA‐PKcs activity and; therefore, increase the resistance of cells to high‐challenge dose radiation‐induced killing. J. Cell. Physiol. 226: 369–374, 2011. © 2010 Wiley‐Liss, Inc.     

A model of interactions between radiation-induced oxidative stress, protein and DNA damage in Deinococcus radiodurans

Ionizing radiation triggers oxidative stress, which can have a variety of subtle and profound biological effects. Here we focus on mathematical modeling of potential synergistic interactions between radiation damage to DNA and oxidative stress-induced damage to proteins involved in DNA repair/replication. When sensitive sites on these proteins are attacked by radiation-induced radicals, correct repair of dangerous DNA lesions such as double strand breaks (DSBs) can be compromised. In contrast, if oxidation of important proteins is prevented by strong antioxidant defenses, DNA repair may function more efficiently. These processes probably occur to some extent even at low doses of radiation/oxidative stress, but they are easiest to investigate at high doses, where both DNA and protein damage are extensive. As an example, we use data on survival of Deinococcus radiodurans after high doses (thousands of Gy) of acute and chronic irradiation. Our model of radiogenic oxidative stress is consistent with these data and can potentially be generalized to other organisms and lower radiation doses.