Quantitative protein network monitoring in response to DNA damage

Conventional molecular biology techniques have identified a large number of cell signaling pathways; however, the importance of these pathways often varies, depending on factors such as treatment type, dose, time after treatment, and cell type. Here, we describe a technique using "reverse-phase" protein lysate microarrays (RPAs) to acquire multiple dimensions of information on protein dynamics in response to DNA damage. Whole-cell lysates from three cellular stress treatments (IR, UV, and ADR) were collected at four doses per treatment, and each, in turn, at 10 time points, resulting in a single-slide RPA consisting of 10,240 features, including replicates. The dynamic molecular profile of 18 unique protein species was compared to phenotypic fate by FACS analysis for corresponding stress conditions. Our initial quantitative results in this new platform confirmed that (1) there is clear stress dose-response effect in p53 protein and (2) a comparison of the rates of increase of p21 and Cyclin D3/p53-Ser15 in response to DNA damage may be associated with the pattern of DNA content. This method, offering a quantitative time-course monitoring of protein expression levels, can provide an experimental reference for developing mathematical models of cell signaling dynamics. Although the present study focuses on the DNA damage-repair pathway, the technique is generally useful to the study of protein signaling.     

Retroviral DNA integration and the DNA damage response

Retroviral DNA integration creates a discontinuity in the host cell chromatin and repair of this damage is required to complete the integration process. As integration and repair are essential for both viral replication and cell survival, it is possible that specific interactions with the host DNA repair systems might provide new cellular targets for human immunodeficiency virus therapy. Various genetic, pharmacological, and biochemical studies have provided strong evidence that postintegration DNA repair depends on components of the nonhomologous end-joining (NHEJ) pathway (DNA-PK (DNA-dependent protein kinase), Ku, Xrcc4, DNA ligase IV) and DNA damage-sensing pathways (Atr (Atm and Rad related), gamma-H2AX). Furthermore, deficiencies in NHEJ components result in susceptibility to apoptotic cell death following retroviral infection. Here, we review these findings and discuss other ways that retroviral DNA intermediates may interact with the host DNA damage signaling and repair pathways.     

Ubiquitin and the DNA damage response

Comment on: Gatti M, et al. Cell Cycle 2012; 11:2538-44.     

Chromatin and the DNA damage response

The impact of chromatin structure upon the DNA damage response is becoming increasingly apparent. We can reasonably expect many more papers showing how chromatin and chromatin modifications impact upon aspects of the DNA damage response. Here, we present our perspective on some recent developments in this exciting area of cell biology. We aim that this review will be of interest to those who study the DNA damage response, but not usually in the context of chromatin, and equally to those who study chromatin, but not the DNA damage response. It seems likely that these two communities will increasingly share common questions and interests.     

组氨酸磷酸化修饰的鉴定方法及应用

蛋白质磷酸化是广受关注的翻译后修饰类型之一,组氨酸磷酸化作为一种非常见的磷酸化修饰,最早被发现在细菌和低等真核生物信号传导的级联反应中起关键作用.近年来研究显示,其在肿瘤发生发展过程中也可能扮演了重要角色.由于磷酸化组氨酸的化学不稳定性、低丰度、亚化学计量性质、缺乏特异性的富集试剂,导致研究手段缺乏,限制了人们对磷酸化...     

Engineering a DNA damage response without DNA damage

Recent work has achieved the feat of activating the DNA damage checkpoint in the absence of DNA damage, revealing the importance of protein-chromatin associations for the activation, amplification and maintenance of the DNA damage response.     

DNA damage response pathway in radioadaptive response

Radioadaptive response is a biological defense mechanism in which low-dose ionizing irradiation elicits cellular resistance to the genotoxic effects of subsequent irradiation. However, its molecular mechanism remains largely unknown. We previously demonstrated that the dose recognition and adaptive response could be mediated by a feedback signaling pathway involving protein kinase C (PKC), p38 mitogen activated protein kinase (p38MAPK) and phospholipase C (PLC). Further, to elucidate the downstream effector pathway, we studied the X-ray-induced adaptive response in cultured mouse and human cells with different genetic background relevant to the DNA damage response pathway, such as deficiencies in TP53, DNA-PKcs, ATM and FANCA genes. The results showed that p53 protein played a key role in the adaptive response while DNA-PKcs, ATM and FANCA were not responsible. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), mimicked the priming irradiation in that the inhibitor alone rendered the cells resistant against the induction of chromosome aberrations and apoptosis by the subsequent X-ray irradiation. The adaptive response, whether it was afforded by low-dose X-rays or wortmannin, occurred in parallel with the reduction of apoptotic cell death by challenging doses. The inhibitor of p38MAPK which blocks the adaptive response did not suppress apoptosis. These observations indicate that the adaptive response and apoptotic cell death constitute a complementary defense system via life-or-death decisions. The p53 has a pivotal role in channeling the radiation-induced DNA double-strand breaks (DSBs) into an adaptive legitimate repair pathway, where the signals are integrated into p53 by a circuitous PKC-p38MAPK-PLC damage sensing pathway, and hence turning off the signals to an alternative pathway to illegitimate repair and apoptosis. A possible molecular mechanism of adaptive response to low-dose ionizing irradiation has been discussed in relation to the repair of DSBs and implicated to the current controversial observations on the expression of adaptive response.     

DNA损伤检测技术

检测DNA损伤的方法有很多,根据其原理大致可以分为3类：基于损伤DNA理化性质的改变检测DNA损伤、基于分子杂交检测DNA损伤以及基于DNA损伤后形成的产物检测DNA损伤。检测DNA损伤的方法目前还在不断快速发展、完善中。本文就DNA损伤的检测方法及其发展做一综述。     

The DNA damage response: sensing and signaling

The protein kinases ATM and ATR are central components of the checkpoint mechanisms that signal the presence of damaged DNA and stalled replication forks. Recent studies have provided important new insights into how these kinases work together with their regulatory subunits, DNA repair proteins and adaptor proteins to sense abnormal DNA structures and implement the appropriate DNA damage response. These advances have provided a more detailed understanding of the interface between damaged DNA and the checkpoint sensor proteins.     

Chromatin modulation and the DNA damage response

The ability to sense and respond appropriately to genetic lesions is vitally important to maintain the integrity of the genome. Emerging evidence indicates that various modulations to chromatin structure are centrally important to many aspects of the DNA damage response (DDR). Here, we discuss recently described roles for specific post-translational covalent modifications to histone proteins, as well as ATP-dependent chromatin remodelling, in DNA damage signalling and repair of DNA double strand breaks.     

Complicated tails: histone modifications and the DNA damage response

In recent years, several ATP-dependent chromatin-remodeling complexes and covalent histone modifications have been implicated in the response to double-stranded DNA breaks (DSBs). When a DSB occurs, cells must identify the DSB, activate the DNA damage checkpoint, and repair the break. Chromatin modification appears to be important but not essential for each of these processes, yet its precise mechanistic roles are only beginning to come into focus. Here, we discuss the role of chromatin in signaling by the DNA damage checkpoint pathway.     

Histone modifications in DNA damage response

DNA damage is a relatively common event in eukaryotic cell and may lead to genetic mutation and even cancer. DNA damage induces cellular responses that enable the cell either to repair the damaged DNA or cope with the damage in an appropriate way. Histone proteins are also the fundamental building blocks of eukaryotic chromatin besides DNA, and many types of post-translational modifications often occur on tails of histones. Although the function of these modifications has remained elusive, there is ever-growing studies suggest that histone modifications play vital roles in several chromatin-based processes, such as DNA damage response. In this review, we will discuss the main histone modifications, and their functions in DNA damage response.     

Redundancy in response to DNA damage

Comment on: Ciznadija D, et al. Cell Cycle 2011; 10:2714-23.     

Competition effect in DNA damage response

We have built an ion-microbeam for studies of the nuclear topography and kinetics of double-strand break repair at the single cell level. Here, we show that a first and a second, delayed single ion exposure at different nuclear sites led to comparable accumulations of phospho-ATM, γ-H2AX and Mdc1 at both earlier (e) and later (l) microirradiated sites. In contrast, accumulations of 53BP1 and the recombination protein Rad51 were strongly reduced at l-sites. This apparent competition effect is accompanied by a reduced amount of 53BP1 in undamaged areas of the irradiated nuclei. We suggest that a critically limited pool size combined with strong binding at irradiated sites leads to the exhaustion of unbound factors freely roaming the nuclear space. The undersupply of these factors at l-sites requires in addition a long-lasting binding at e-sites or a weaker binding at l-sites. The observed effects suggest that DNA damage response at individual nuclear sites depends on the time course of damage load. This may have implications for therapeutic radiation treatments. Christoph Greubel, Volker Hable and Guido A. Drexler contributed equally to this work.