DNA double-strand breaks (DSBs) are deleterious DNA lesions that if left unrepaired or are misrepaired, potentially result in chromosomal aberrations, known drivers of carcinogenesis. Pathways that ...direct the repair of DSBs are traditionally believed to be guardians of the genome as they protect cells from genomic instability. The prominent DSB repair pathway in human cells is the non-homologous end joining (NHEJ) pathway, which mediates template-independent re-ligation of the broken DNA molecule and is active in all phases of the cell cycle. Its role as a guardian of the genome is supported by the fact that defects in NHEJ lead to increased sensitivity to agents that induce DSBs and an increased frequency of chromosomal aberrations. Conversely, evidence from tumors and tumor cell lines has emerged that NHEJ also promotes chromosomal aberrations and genomic instability, particularly in cells that have a defect in one of the other DSB repair pathways. Collectively, the data present a conundrum: how can a single pathway both suppress and promote carcinogenesis? In this review, we will examine NHEJ's role as both a guardian and a disruptor of the genome and explain how underlying genetic context not only dictates whether NHEJ promotes or suppresses carcinogenesis, but also how it alters the response of tumors to conventional therapeutics.
Abstract
The DNA damage response (DDR) encompasses the cellular response to DNA double-stranded breaks (DSBs), and includes recognition of the DSB, recruitment of numerous factors to the DNA damage ...site, initiation of signaling cascades, chromatin remodeling, cell-cycle checkpoint activation, and repair of the DSB. Key drivers of the DDR are multiple members of the phosphatidylinositol 3-kinase-related kinase family, including ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). ATM and ATR modulate multiple portions of the DDR, but DNA-PKcs is believed to primarily function in the DSB repair pathway, non-homologous end joining. Utilizing a human cell line in which the kinase domain of DNA-PKcs is inactivated, we show here that DNA-PKcs kinase activity is required for the cellular response to DSBs immediately after their induction. Specifically, DNA-PKcs kinase activity initiates phosphorylation of the chromatin factors H2AX and KAP1 following ionizing radiation exposure and drives local chromatin decondensation near the DSB site. Furthermore, loss of DNA-PKcs kinase activity results in a marked decrease in the recruitment of numerous members of the DDR machinery to DSBs. Collectively, these results provide clear evidence that DNA-PKcs activity is pivotal for the initiation of the DDR.
Viscous Marangoni propulsion Lauga, Eric; Davis, Anthony M. J.
Journal of fluid mechanics,
08/2012, Letnik:
705
Journal Article
Recenzirano
Odprti dostop
Marangoni propulsion is a form of locomotion wherein an asymmetric release of surfactant by a body located at the surface of a liquid leads to its directed motion. We present in this paper a ...mathematical model for Marangoni propulsion in the viscous regime. We consider the case of a thin rigid circular disk placed at the surface of a viscous fluid and whose perimeter has a prescribed concentration of an insoluble surfactant, to which the rest of its surface is impenetrable. Assuming a linearized equation of state between surface tension and surfactant concentration, we derive analytically the surfactant, velocity and pressure fields in the asymptotic limit of low capillary, Péclet and Reynolds numbers. We then exploit these results to calculate the Marangoni propulsion speed of the disk. Neglecting the stress contribution from Marangoni flows is seen to over-predict the propulsion speed by 50 %.
RecQ DNA helicases are a conserved protein family found in bacteria, fungus, plants, and animals. These helicases play important roles in multiple cellular functions, including DNA replication, ...transcription, DNA repair, and telomere maintenance. Humans have five RecQ helicases: RECQL1, Bloom syndrome protein (BLM), Werner syndrome helicase (WRN), RECQL4, and RECQL5. Defects in BLM and WRN cause autosomal disorders: Bloom syndrome (BS) and Werner syndrome (WS), respectively. Mutations in RECQL4 are associated with three genetic disorders, Rothmund-Thomson syndrome (RTS), Baller-Gerold syndrome (BGS), and RAPADILINO syndrome. Although no genetic disorders have been reported due to loss of RECQL1 or RECQL5, dysfunction of either gene is associated with tumorigenesis. Multiple genetically independent pathways have evolved that mediate the repair of DNA double-strand break (DSB), and RecQ helicases play pivotal roles in each of them. The importance of DSB repair is supported by the observations that defective DSB repair can cause chromosomal aberrations, genomic instability, senescence, or cell death, which ultimately can lead to premature aging, neurodegeneration, or tumorigenesis. In this review, we will introduce the human RecQ helicase family, describe in detail their roles in DSB repair, and provide relevance between the dysfunction of RecQ helicases and human diseases.
Ataxia telangiectasia mutated (ATM) plays a critical role in the cellular response to DNA damage. In response to DNA double-strand breaks (DSBs), ATM is autophosphorylated at serine 1981. Although ...this autophosphorylation is widely considered a sign of ATM activation, it is still not clear if autophosphorylation is required for ATM functions including localization to DSBs and activation of ATM kinase activity. In this study, we show that localization of ATM to DSBs is differentially regulated with the initial localization requiring the MRE11-RAD50-NBS1 complex and sustained retention requiring autophosphorylation of ATM at serine 1981. Autophosphorylated ATM interacts with MDC1 and the latter is required for the prolonged association of ATM to DSBs. Ablation of ATM autophosphorylation or knock-down of MDC1 protein affects the ability of ATM to phosphorylate downstream substrates and confer radioresistance. Together, these data suggest that autophosphorylation at serine 1981 stabilizes ATM at the sites of DSBs, and this is required for a proper DNA damage response.
Abstract
Non-homologous end joining (NHEJ) is the major pathway that mediates the repair of DNA double-strand breaks (DSBs) generated by ionizing radiation (IR). Previously, the DNA helicase RECQL4 ...was implicated in promoting NHEJ, but its role in the pathway remains unresolved. In this study, we report that RECQL4 stabilizes the NHEJ machinery at DSBs to promote repair. Specifically, we find that RECQL4 interacts with the NHEJ core factor DNA-PKcs and the interaction is increased following IR. RECQL4 promotes DNA end bridging mediated by DNA-PKcs and Ku70/80 in vitro and the accumulation/retention of NHEJ factors at DSBs in vivo. Moreover, interaction between DNA-PKcs and the other core NHEJ proteins following IR treatment is attenuated in the absence of RECQL4. These data indicate that RECQL4 promotes the stabilization of the NHEJ factors at DSBs to support formation of the NHEJ long-range synaptic complex. In addition, we observed that the kinase activity of DNA-PKcs is required for accumulation of RECQL4 to DSBs and that DNA-PKcs phosphorylates RECQL4 at six serine/threonine residues. Blocking phosphorylation at these sites reduced the recruitment of RECQL4 to DSBs, attenuated the interaction between RECQL4 and NHEJ factors, destabilized interactions between the NHEJ machinery, and resulted in decreased NHEJ. Collectively, these data illustrate reciprocal regulation between RECQL4 and DNA-PKcs in NHEJ.
Abstract
Ataxia-telangiectasia mutated (ATM) drives the DNA damage response via modulation of multiple signal transduction and DNA repair pathways. Previously, ATM activity was implicated in ...promoting the non-homologous end joining (NHEJ) pathway to repair a subset of DNA double-stranded breaks (DSBs), but how ATM performs this function is still unclear. In this study, we identified that ATM phosphorylates the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a core NHEJ factor, at its extreme C-terminus at threonine 4102 (T4102) in response to DSBs. Ablating phosphorylation at T4102 attenuates DNA-PKcs kinase activity and this destabilizes the interaction between DNA-PKcs and the Ku-DNA complex, resulting in decreased assembly and stabilization of the NHEJ machinery at DSBs. Phosphorylation at T4102 promotes NHEJ, radioresistance, and increases genomic stability following DSB induction. Collectively, these findings establish a key role for ATM in NHEJ-dependent repair of DSBs through positive regulation of DNA-PKcs.
Graphical Abstract
Graphical Abstract
Phosphorylation of DNA-PKcs at T4102 by ATM promotes NHEJ-mediated DSB repair.
Pathway choice within DNA double-strand break (DSB) repair is a tightly regulated process to maintain genome integrity. RECQL4, deficient in Rothmund-Thomson Syndrome, promotes the two major DSB ...repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). Here we report that RECQL4 promotes and coordinates NHEJ and HR in different cell cycle phases. RECQL4 interacts with Ku70 to promote NHEJ in G1 when overall cyclin-dependent kinase (CDK) activity is low. During S/G2 phases, CDK1 and CDK2 (CDK1/2) phosphorylate RECQL4 on serines 89 and 251, enhancing MRE11/RECQL4 interaction and RECQL4 recruitment to DSBs. After phosphorylation, RECQL4 is ubiquitinated by the DDB1-CUL4A E3 ubiquitin ligase, which facilitates its accumulation at DSBs. Phosphorylation of RECQL4 stimulates its helicase activity, promotes DNA end resection, increases HR and cell survival after ionizing radiation, and prevents cellular senescence. Collectively, we propose that RECQL4 modulates the pathway choice of NHEJ and HR in a cell cycle-dependent manner.
Abstract
Multiple pathways mediate the repair of DNA double-strand breaks (DSBs), with numerous mechanisms responsible for driving choice between the pathways. Previously, we reported that mutating ...five putative phosphorylation sites on the non-homologous end joining (NHEJ) factor, Ku70, results in sustained retention of human Ku70/80 at DSB ends and attenuation of DSB repair via homologous recombination (HR). In this study, we generated a knock-in mouse, in which the three conserved putative phosphorylation sites of Ku70 were mutated to alanine to ablate potential phosphorylation (Ku703A/3A), in order to examine if disrupting DSB repair pathway choice by modulating Ku70/80 dynamics at DSB ends results in enhanced genomic instability and tumorigenesis. The Ku703A/3A mice developed spontaneous and have accelerated chemical-induced hepatocellular carcinoma (HCC) compared to wild-type (Ku70+/+) littermates. The HCC tumors from the Ku703A/3A mice have increased γH2AX and 8-oxo-G staining, suggesting decreased DNA repair. Spontaneous transformed cell lines from Ku703A/3A mice are more radiosensitive, have a significant decrease in DNA end resection, and are more sensitive to the DNA cross-linking agent mitomycin C compared to cells from Ku70+/+ littermates. Collectively, these findings demonstrate that mutating the putative Ku70 phosphorylation sites results in defective DNA damage repair and disruption of this process drives genomic instability and accelerated development of HCC.
Graphical Abstract
Graphical Abstract
Mutating the putative Ku70 phosphorylation sites in a mouse model results in decreased DNA repair capacity, which induces chronic DNA damage and genomic instability and this subsequently drives the induction of spontaneous hepatocellular carcinoma. Graphical Abstract was created with BioRender.com.
Compared to conventional photon-based external beam radiation (PhXRT), carbon ion radiotherapy (CIRT) has superior dose distribution, higher linear energy transfer (LET), and a higher relative ...biological effectiveness (RBE). This enhanced RBE is driven by a unique DNA damage signature characterized by clustered lesions that overwhelm the DNA repair capacity of malignant cells. These physical and radiobiological characteristics imbue heavy ions with potent tumoricidal capacity, while having the potential for simultaneously maximally sparing normal tissues. Thus, CIRT could potentially be used to treat some of the most difficult to treat tumors, including those that are hypoxic, radio-resistant, or deep-seated. Clinical data, mostly from Japan and Germany, are promising, with favorable oncologic outcomes and acceptable toxicity. In this manuscript, we review the physical and biological rationales for CIRT, with an emphasis on DNA damage and repair, as well as providing a comprehensive overview of the translational and clinical data using CIRT.
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