DNA polymerase (Pol) is an error-prone DNA polymerase involved in translesion DNA synthesis. Pol consists of two subunits: the catalytic REV3, which belongs to B family DNA polymerase, and the noncatalytic REV7. REV7 also interacts with REV1 polymerase, which is an error-prone Y family DNA polymerase and is also involved in translesion DNA synthesis. Cells deficient in one of the three REV proteins and those deficient in all three proteins show similar phenotype, indicating the functional collaboration of the three REV proteins. REV7 interacts with both REV3 and REV1 polymerases, but the structure of REV7 or REV3, as well as the structural and functional basis of the REV1-REV7 and REV3-REV7 interactions, remains unknown. Here we show the first crystal structure of human REV7 in complex with a fragment of human REV3 polymerase (residues 1847-1898) and reveal the mechanism underlying REV7-REV3 interaction. The structure indicates that the interaction between REV7 and REV3 creates a structural interface for REV1 binding. Furthermore, we show that the REV7-mediated interactions are responsible for DNA damage tolerance. Our results highlight the function of REV7 as an adapter protein to recruit Pol to a lesion site. REV7 is alternatively called MAD2B or MAD2L2 and also involved in various cellular functions such as signal transduction and cell cycle regulation. Our results will provide a general structural basis for understanding the REV7 interaction.Large numbers of DNA lesions occur daily in every cell, and the majority of the DNA lesions stall replicative DNA polymerases. This results in the arrest of DNA replication, which causes lethal effects including genome instability and cell death. Translesion DNA synthesis (TLS)2 releases this replication blockage by replacing the stalled replicative polymerase with a DNA polymerase specialized for TLS (TLS polymerase). It is generally considered that TLS includes two steps performed by at least two types of TLS polymerases, namely inserter and extender polymerases (reviewed in Refs. 1 and 2). In the first step, the stalled replicative polymerase is switched to an inserter polymerase such as Pol, Pol, Pol, or REV1, which are classified as Y family DNA polymerases (3) and have different lesion specificity (reviewed in Refs. 4 -8), and an inserter polymerase incorporates nucleotides opposite the DNA lesion instead of the stalled replicative polymerase. In the second step, an inserter polymerase is switched to the extender polymerase DNA polymerase (Pol), and then Pol extends a few additional nucleotides before a replicative polymerase restarts DNA replication.Pol consists of the catalytic REV3 and the noncatalytic REV7 subunits. REV3 is classified as a B family DNA polymerase on the basis of the primary sequence. The catalytic activity of yeast REV3 is stimulated by yeast REV7 (9). Biochemical analysis has been done only for yeast REV3 but not mammalian REV3, because the molecular mass of human REV3 is larger (ϳ350 kDa) than that of yeast REV3 (ϳ150 kDa). Disruption of the ...
Interstrand cross-links (ICLs) block replication and transcription and thus are highly cytotoxic. In higher eukaryotes, ICLs processing involves the Fanconi anemia (FA) pathway and homologous recombination. Stalled replication forks activate the eight-subunit FA core complex, which ubiquitylates FANCD2-FANCI. Once it is posttranslationally modified, this heterodimer recruits downstream members of the ICL repairosome, including the FAN1 nuclease. However, ICL processing has been shown to also involve MUS81-EME1 and XPF-ERCC1, nucleases known to interact with SLX4, a docking protein that also can bind another nuclease, SLX1. To investigate the role of SLX4 more closely, we disrupted the SLX4 gene in avian DT40 cells. SLX4 deficiency caused cell death associated with extensive chromosomal aberrations, including a significant fraction of isochromatid-type breaks, with sister chromatids broken at the same site. SLX4 thus appears to play an essential role in cell proliferation, probably by promoting the resolution of interchromatid homologous recombination intermediates. Because ubiquitylation plays a key role in the FA pathway, and because the N-terminal region of SLX4 contains a ubiquitinbinding zinc finger (UBZ) domain, we asked whether this domain is required for ICL processing. We found that SLX4 −/− cells expressing UBZ-deficient SLX4 were selectively sensitive to ICL-inducing agents, and that the UBZ domain was required for interaction of SLX4 with ubiquitylated FANCD2 and for its recruitment to DNAdamage foci generated by ICL-inducing agents. Our findings thus suggest that ubiquitylated FANCD2 recruits SLX4 to DNA damage sites, where it mediates the resolution of recombination intermediates generated during the processing of ICLs.endonuclease | mitomycin C | cisplatin | DNA repair I nterstrand cross-links (ICLs) inhibit transcription and replication. Their considerable cytotoxicity has been ascribed primarily to their blockage of replication forks, and this phenomenon is believed to be responsible for the success of ICL-inducing agents, such as cisplatin and mitomycin-C (MMC), in cancer chemotherapy (1). ICL processing is complex, involving proteins from several distinct pathways of DNA metabolism. In higher organisms, ICL processing is orchestrated by the Fanconi anemia (FA) pathway (2, 3). Collision of replication forks with ICLs activates the ATR kinase, which in turn licenses the FANCL ubiquitin ligase subunit of the FA core complex (composed of FANCA, B, C, E, F, G, L and M proteins) to modify the FANCD2-FANCI heterodimer (2, 4-6). The monoubiquitylated FANCD2-FANCI complex is then targeted to chromatin (7,8), where it recruits downstream components of the repairosome, including the structure-specific nuclease FAN1 (9-12). However, ICL processing also requires other enzymes, such as the nucleases MUS81-EME1 and XPF-ERCC1, and how these are recruited to sites of damage in ICL repair is not known.The structure-specific endonucleases XPF-ERCC1 and MUS81-EME1, which are implicated in ICL repair and in the res...
The lithium ion phase-transfer reaction between the spinel lithium manganese oxide electrode and a nonaqueous electrolyte was investigated by the ac impedance spectroscopic method. The dependence of the impedance spectra on the electrochemical potential of the lithium ion in the electrode, the lithium salt concentration in the electrolyte, the kind of solvent, and the measured temperature were examined. Nyquist plots, obtained from the impedance measurements, consist of two semicircles for high and medium frequency and warburg impedance for low frequency, indicating that the reaction process of two main steps for high and medium frequency obey the Butler-Volmer type equation and could be related to the charge-transfer reaction process accompanied with lithium ion phase-transfer at the interface. The dependency on the solvent suggests that both steps in the lithium ion phase-transfer at the electrode/electrolyte interface include the desolvation process and have high activation barriers.
ABSTRACT:Primary hepatocytes have been used in drug development for the evaluation of hepatotoxicity of candidate compounds. However, the rapid depression of their hepatic characters in vitro must be improved to predict toxicity with higher accuracy. We have hypothesized that a well organized tissue construct that includes nonparenchymal cells and appropriate scaffold material(s) could overcome this difficulty by remediating the viability and physiological function of primary hepatocytes. In this study, we constructed an in vitro liver tissue model, consisting of mouse primary hepatocytes assembling around an endothelial cell network on Engelbreth-Holm-Swarm gel, and examined its response to acetaminophen treatment. The increase in lactate dehydrogenase release after the exposure to acetaminophen was induced earlier in the liver tissue model than in monolayer hepatocytes alone, suggesting that the tissue model was more sensitive to an acetaminopheninduced toxicity. On the basis of our results, we conclude that liver tissue models of this kind may enhance the responses of hepatocytes against xenobiotics via the maintenance of hepatic genes and functions such as cytochrome P450s. These findings will contribute to the development of more accurate systems for evaluating hepatotoxicity.
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