Dynamics of Chromatin during the Repair of DNA Double-Strand Breaks

Double strand breaks (DSBs) are arguably the most deleterious DNAlesion that a cell can sustain, and defects in the ability to detect and repair thesebreaks result in increased genomic instability and have been causatively linked tocancer. The repair of DNA DSBs must occur in the context of chromatin, andthere is increasing evidence that the modulation of chromatin plays an integralrole in the DNA DSB repair process. Here, we summarize a number of keyfindings, largely from studies performed in budding yeast, highlighting the role ofchromatin in DNA DSB responses.     

Molecular Mechanism of Allosteric Communication in Hsp70 Revealed by Molecular Dynamics Simulations

Investigating ligand-regulated allosteric coupling between protein domains is fundamental to understand cell-life regulation. The Hsp70 family of chaperones represents an example of proteins in which ATP binding and hydrolysis at the Nucleotide Binding Domain (NBD) modulate substrate recognition at the Substrate Binding Domain (SBD). Herein, a comparative analysis of an allosteric (Hsp70-DnaK) and a non-allosteric structural homolog (Hsp110-Sse1) of the Hsp70 family is carried out through molecular dynamics simulations, starting from different conformations and ligand-states. Analysis of ligand-dependent modulation of internal fluctuations and local deformation patterns highlights the structural and dynamical changes occurring at residue level upon ATP-ADP exchange, which are connected to the conformational transition between closed and open structures. By identifying the dynamically responsive protein regions and specific cross-domain hydrogen-bonding patterns that differentiate Hsp70 from Hsp110 as a function of the nucleotide, we propose a molecular mechanism for the allosteric signal propagation of the ATP-encoded conformational signal.     

DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living Cells

Chromosome breakage is a major threat to genome integrity. The most accurate way to repair DNA double strand breaks (DSB) is homologous recombination (HR) with an intact copy of the broken locus. Mobility of the broken DNA has been seen to increase during the search for a donor copy. Observing chromosome dynamics during the earlier steps of HR, mainly the resection from DSB ends that generates recombinogenic single strands, requires a visualization system that does not interfere with the process, and is small relative to the few kilobases of DNA that undergo processing. Current visualization tools, based on binding of fluorescent repressor proteins to arrays of specific binding sites, have the major drawback that highly-repeated DNA and lengthy stretches of strongly bound protein can obstruct chromatin function. We have developed a new, non-intrusive method which uses protein oligomerization rather than operator multiplicity to form visible foci. By applying it to HO cleavage of the MAT locus on Saccharomyces cerevisiae chromosome III, we provide the first real-time analysis of resection in single living cells. Monitoring the dynamics of a chromatin locus next to a DSB revealed transient confinement of the damaged chromatin region during the very early steps of resection, consistent with the need to keep DNA ends in contact. Resection in a yku70 mutant began ∼10 min earlier than in wild type, defining this as the period of commitment to homology-dependent repair. Beyond the insights into the dynamics and mechanism of resection, our new DNA-labelling and -targeting method will be widely applicable to fine-scale analysis of genome organization, dynamics and function in normal and pathological contexts.     

Spatio-Temporal Dynamics of Chromatin Containing DNA Breaks

The cellular response to DNA breaks consists of a complex signaling network that coordinates the initial recognition of the lesion with the induction of cell cycle checkpoints and DNA repair. With DNA wrapped around histone proteins and packaged into higher order levels of chromatin structure, the detection of a single DNA break (DSB) in the genome is the molecular equivalent of finding a needle in a haystack . A recent study from our laboratory used high-resolution electron microscropy and live cell imaging to demonstrate that chromatin undergoes a marked reorganization in response to a DSB. In an energy dependent manner, chromatin rapidly decondenses to a more open configuration in the regions surrounding the lesion. We propose that this ATP dependent chromatin remodeling event facilitates the subsequent recognition and processing of damaged DNA. While the chromatin surrounding the lesion remodels to a more open configuration, the DNA break itself remains relatively immobile over time, consistent with the idea that DNA damage response proteins migrate to positionally stable sites of damaged DNA 1. The lack of significant movement of chromatin regions containing DSBs has implications for the process by which chromosomal translocations form.     

The Gating Mechanism of the Human Aquaporin 5 Revealed by Molecular Dynamics Simulations

Aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). The high-resolution X-ray structure of the human aquaporin 5 (HsAQP5) shows that HsAQP5, as all the other known aquaporins, exhibits tetrameric structure. By means of molecular dynamics simulations we analyzed the role of spontaneous fluctuations on the structural behavior of the human AQP5. We found that different conformations within the tetramer lead to a distribution of monomeric channel structures, which can be characterized as open or closed. The switch between the two states of a channel is a tap-like mechanism at the cytoplasmic end which regulates the water passage through the pore. The channel is closed by a translation of the His67 residue inside the pore. Moreover, water permeation rate calculations revealed that the selectivity filter, located at the other end of the channel, regulates the flow rate of water molecules when the channel is open, by locally modifying the orientation of His173. Furthermore, the calculated permeation rates of a fully open channel are in good agreement with the reported experimental value.     

A Model of Lipid-Free Apolipoprotein A-I Revealed by Iterative Molecular Dynamics Simulation

Apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, has been proven inversely correlated to cardiovascular risk in past decades. The lipid-free state of apo A-I is the initial stage which binds to lipids forming high-density lipoprotein. Molecular models of lipid-free apo A-I have been reported by methods like X-ray crystallography and chemical cross-linking/mass spectrometry (CCL/MS). Through structural analysis we found that those current models had limited consistency with other experimental results, such as those from hydrogen exchange with mass spectrometry. Through molecular dynamics simulations, we also found those models could not reach a stable equilibrium state. Therefore, by integrating various experimental results, we proposed a new structural model for lipid-free apo A-I, which contains a bundled four-helix N-terminal domain (1–192) that forms a variable hydrophobic groove and a mobile short hairpin C-terminal domain (193–243). This model exhibits an equilibrium state through molecular dynamics simulation and is consistent with most of the experimental results known from CCL/MS on lysine pairs, fluorescence resonance energy transfer and hydrogen exchange. This solution-state lipid-free apo A-I model may elucidate the possible conformational transitions of apo A-I binding with lipids in high-density lipoprotein formation.     

Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

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A Possible Binding Path of Ergosterol Within Elicitins Revealed by Molecular Dynamics

Abstract

Elicitins, produced by most of the phytopathogenic fungi of the genus Phytophthora, provoke in the tobacco plant both remote leaf necrosis and the induction of a resistance against subsequent attack by various micro-organisms. The crystal structure of b-cryptogein (CRY), secreted by Phytophthora cryptogea, was previously reported as well as the first structure of a SCP/sterol complex, the ergosterol-complexed, mutated CRY (K13H). In K13H, the ergosterol molecule is encapsulated in a large internal hydrophobic cavity which is not present in CRY. This binding induces a minor conformational change in the protein structure. Molecular dynamics studies were undertaken to precise the structural behaviour of CRY and K13H with respect to the complexation of the ergosterol.

Although it is not possible to simulate the entrance of the ergosterol in the protein, we assume that capture and release of the ligand possibly both occur following the same path. Our results show that, in the complex K13H, the ergosterol molecule is pushed towards the residue 13 which play a key role in the necrotic activity of the protein. It is likely that the polarity of residue 13, favouring the binding of the hydroxy l of the ligand, would be involved in the recognition of the sterol and in an optimisation of its orientation. Thus, in a first step, the molecule of ergosterol would be rotated around itself to a position which makes possible, in a second step, its translation to the internal cavity, as a key in a keyhole.     

表观遗传调控：基于染色质环境的DNA双链断裂修复

DNA双链断裂（DNA double-strand breaks, DSBs）是威胁基因组完整性和细胞存活的最有害的DNA损伤类型。同源重组（homologous recombination,HR）和非同源末端连接（non-homologous end joining,NHEJ）是修复DNA双链断裂的两种主要途径。DSB修复涉及到损伤部位修复蛋白的募集和染色质结构的改变。在DNA双链断裂诱导下,染色质结构的动态变化在时间和空间上受到严格调控,进而对DNA双链断裂修复过程进行精细调节。特定的染色质修饰形成利于修复的染色质状态,有助于DNA双链断裂修复机器的招募、修复途径的选择和DNA损伤检查点的活化;其中修复途径的选择对于基因组稳定性至关重要。修复不当或失败可导致基因组不稳定性,甚至促进肿瘤的发生。本文综述了染色质结构和染色质修饰的动态变化在DSB修复中的重要作用。此外,文章还总结了在癌症治疗中靶向关键染色质调控因子在基因组稳定性维持、肿瘤发生发展以及潜在临床应用价值等方面的进展。     

DNA修复的分子机制

ＤＮＡ是遗传信息的载体,需要有极高的保真度,这不仅有赖于完善的复制体系,而且还需要有能纠正已存在错误的修复系统。对于不同的ＤＮＡ损伤,生物体内存在许多不同的修复系统。本文介绍三种主要修复系统即核苷酸切割修复,错配修复及转录偶联修复的分子机制,深入研究ＤＮＡ修复作用对了解某些癌症成因及细胞衰老等过程有重要意义 。     

Ligand Induced Conformational Changes of the Human Serotonin Transporter Revealed by Molecular Dynamics Simulations

The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design.