Lipid peroxidation product 4-hydroxy-2-nonenal modulates base excision repair in human cells

Oxidative-stress-driven lipid peroxidation (LPO) is involved in the pathogenesis of several human diseases, including cancer. LPO products react with cellular proteins changing their properties, and with DNA bases to form mutagenic etheno-DNA adducts, removed from DNA mainly by the base excision repair (BER) pathway.One of the major reactive aldehydes generated by LPO is 4-hydroxy-2-nonenal (HNE). We investigated the effect of HNE on BER enzymes in human cells and in vitro. K21 cells pretreated with physiological HNE concentrations were more sensitive to oxidative and alkylating agents, H₂O₂ and MMS, than were untreated cells. Detailed examination of the effects of HNE on particular stages of BER in K21 cells revealed that HNE decreases the rate of excision of 1,N⁶-ethenoadenine (ɛA) and 3,N⁴-ethenocytosine (ɛC), but not of 8-oxoguanine. Simultaneously HNE increased the rate of AP-site incision and blocked the re-ligation step after the gap-filling by DNA polymerases. This suggested that HNE increases the number of unrepaired single-strand breaks (SSBs) in cells treated with oxidizing or methylating agents. Indeed, preincubation of cells with HNE and their subsequent treatment with H₂O₂ or MMS increased the number of nuclear poly(ADP-ribose) foci, known to appear in cells in response to SSBs. However, when purified BER enzymes were exposed to HNE, only ANPG and TDG glycosylases excising ɛA and ɛC from DNA were inhibited, and only at high HNE concentrations. APE1 endonuclease and 8-oxoG-DNA glycosylase 1 (OGG1) were not inhibited. These results indicate that LPO products exert their promutagenic action not only by forming DNA adducts, but in part also by compromising the BER pathway.     

Inorganic arsenic inhibits the nucleotide excision repair pathway and reduces the expression of XPC

Chronic exposure to arsenic, most often through contaminated drinking water, has been linked to several types of cancer in humans, including skin and lung cancer. However, the mechanisms underlying its role in causing cancer are not well understood. There is evidence that exposure to arsenic can enhance the carcinogenicity of UV light in inducing skin cancers and may enhance the carcinogenicity of tobacco smoke in inducing lung cancers. The nucleotide excision repair (NER) pathway removes different types of DNA damage including those produced by UV light and components of tobacco smoke. The aim of the present study was to investigate the effect of sodium arsenite on the NER pathway in human lung fibroblasts (IMR-90 cells) and primary mouse keratinocytes. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6-4 photoproducts (6-4 PP) and cyclobutane pyrimidine dimers (CPDs). We find a concentration-dependent inhibition of the removal of 6-4 PPs and CPDs in both cell types treated with arsenite. Treatment of both cell types with arsenite resulted in a significant reduction in the abundance of XPC, a protein that is critical for DNA damage recognition in NER. The abundance of RNA expressed from several key NER genes was also significantly reduced by treatment of IMR-90 cells with arsenite. Finally, treatment of IMR-90 cells with MG-132 abrogated the reduction in XPC protein, suggesting an involvement of the proteasome in the reduction of XPC protein produced by treatment of cells with arsenic. The inhibition of NER by arsenic may reflect one mechanism underlying the role of arsenic exposure in enhancing cigarette smoke-induced lung carcinogenesis and UV light-induced skin cancer, and it may provide some insights into the emergence of arsenic trioxide as a chemotherapeutic agent.     

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

BRCA1: Beyond double-strand break repair

Since its discovery, the BRCA1 tumor suppressor has been shown to play a role in multiple DNA damage response pathways. Here, we will review the involvement of BRCA1 in base-excision DNA repair and highlight its clinical implications.     

DR-GAS: A database of functional genetic variants and their phosphorylation states in human DNA repair systems

We present DR-GAS¹, a unique, consolidated and comprehensive DNA repair genetic association studies database of human DNA repair system. It presents information on repair genes, assorted mechanisms of DNA repair, linkage disequilibrium, haplotype blocks, nsSNPs, phosphorylation sites, associated diseases, and pathways involved in repair systems. DNA repair is an intricate process which plays an essential role in maintaining the integrity of the genome by eradicating the damaging effect of internal and external changes in the genome. Hence, it is crucial to extensively understand the intact process of DNA repair, genes involved, non-synonymous SNPs which perhaps affect the function, phosphorylated residues and other related genetic parameters. All the corresponding entries for DNA repair genes, such as proteins, OMIM IDs, literature references and pathways are cross-referenced to their respective primary databases. DNA repair genes and their associated parameters are either represented in tabular or in graphical form through images elucidated by computational and statistical analyses. It is believed that the database will assist molecular biologists, biotechnologists, therapeutic developers and other scientific community to encounter biologically meaningful information, and meticulous contribution of genetic level information towards treacherous diseases in human DNA repair systems. DR-GAS is freely available for academic and research purposes at: http://www.bioinfoindia.org/drgas.     

Glycolysis-mediated control of blood-brain barrier development and function

The blood-brain barrier (BBB) consists of differentiated cells integrating in one ensemble to control transport processes between the central nervous system (CNS) and peripheral blood. Molecular organization of BBB affects the extracellular content and cell metabolism in the CNS. Developmental aspects of BBB attract much attention in recent years, and barriergenesis is currently recognized as a very important and complex mechanism of CNS development and maturation. Metabolic control of angiogenesis/barriergenesis may be provided by glucose utilization within the neurovascular unit (NVU). The role of glycolysis in the brain has been reconsidered recently, and it is recognized now not only as a process active in hypoxic conditions, but also as a mechanism affecting signal transduction, synaptic activity, and brain development. There is growing evidence that glycolysis-derived metabolites, particularly, lactate, affect barriergenesis and functioning of BBB. In the brain, lactate produced in astrocytes or endothelial cells can be transported to the extracellular space via monocarboxylate transporters (MCTs), and may act on the adjoining cells via specific lactate receptors. Astrocytes are one of the major sources of lactate production in the brain and significantly contribute to the regulation of BBB development and functioning. Active glycolysis in astrocytes is required for effective support of neuronal activity and angiogenesis, while endothelial cells regulate bioavailability of lactate for brain cells adjusting its bidirectional transport through the BBB. In this article, we review the current knowledge with regard to energy production in endothelial and astroglial cells within the NVU. In addition, we describe lactate-driven mechanisms and action of alternative products of glucose metabolism affecting BBB structural and functional integrity in developing and mature brain.     

ARTD1 (PARP1) activation and NAD+ in DNA repair and cell death

Nicotinamide adenine dinucleotide, NAD⁺, is a small metabolite coenzyme that is essential for the progress of crucial cellular pathways including glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial respiration. These processes consume and produce both oxidative and reduced forms of NAD (NAD⁺ and NADH). NAD⁺ is also important for ADP(ribosyl)ation reactions mediated by the ADP-ribosyltransferase enzymes (ARTDs) or deacetylation reactions catalyzed by the sirtuins (SIRTs) which use NAD⁺ as a substrate. In this review, we highlight the significance of NAD⁺ catabolism in DNA repair and cell death through its utilization by ARTDs and SIRTs. We summarize the current findings on the involvement of ARTD1 activity in DNA repair and most specifically its involvement in the trigger of cell death mediated by ARTD1 activation and energy depletion. By sharing the same substrate, the activities of ARTDs and SIRTs are tightly linked, are dependent on each other and are thereby involved in the same cellular processes that play an important role in cancer biology, inflammatory diseases and ischaemia/reperfusion.     

Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C_18:1) but lower levels of hexadecanoic (C_16:0) and palmitelaidic (C_16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance.     

A C. elegans homolog of the Cockayne syndrome complementation group A gene

Cockayne syndrome (CS) is a debilitating and complex disorder that results from inherited mutations in the CS complementation genes A and B, CSA and CSB. The links between the molecular functions of the CS genes and the complex pathophysiology of CS are as of yet poorly understood and are the subject of intense debate. While mouse models reflect the complexity of CS, studies on simpler genetic models might shed new light on the consequences of CS mutations. Here we describe a functional homolog of the human CSA gene in Caenorhabditis elegans. Similar to its human counterpart, mutations in the nematode csa-1 gene lead to developmental growth defects as a consequence of DNA lesions.     

ATMIN is required for the ATM-mediated signaling and recruitment of 53BP1 to DNA damage sites upon replication stress

Unresolved replication intermediates can block the progression of replication forks and become converted into DNA lesions, hence exacerbating genomic instability. The p53-binding protein 1 (53BP1) forms nuclear bodies at sites of unrepaired DNA lesions to shield these regions against erosion, in a manner dependent on the DNA damage kinase ATM. The molecular mechanism by which ATM is activated upon replicative stress to localize the 53BP1 protection complex is unknown. Here we show that the ATM-INteracting protein ATMIN (also known as ASCIZ) is partially required for 53BP1 localization upon replicative stress. Additionally, we demonstrate that ATM activation is impaired in cells lacking ATMIN and we define that ATMIN is required for initiating ATM signaling following replicative stress. Furthermore, loss of ATMIN leads to chromosomal segregation defects. Together these data reveal that chromatin integrity depends on ATMIN upon exposure to replication-induced stress.     

RNA-directed repair of DNA double-strand breaks

DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, which if unrepaired or repaired incorrectly can cause cell death or genome instability that may lead to cancer. To counteract these adverse consequences, eukaryotes have evolved a highly orchestrated mechanism to repair DSBs, namely DNA-damage-response (DDR). DDR, as defined specifically in relation to DSBs, consists of multi-layered regulatory modes including DNA damage sensors, transducers and effectors, through which DSBs are sensed and then repaired via DNAprotein interactions. Unexpectedly, recent studies have revealed a direct role of RNA in the repair of DSBs, including DSB-induced small RNA (diRNA)-directed and RNA-templated DNA repair. Here, we summarize the recent discoveries of RNA-mediated regulation of DSB repair and discuss the potential impact of these novel RNA components of the DSB repair pathway on genomic stability and plasticity.     

DNA-PK: A dynamic enzyme in a versatile DSB repair pathway

DNA double stranded breaks (DSBs) are the most cytoxic DNA lesion as the inability to properly repair them can lead to genomic instability and tumorigenesis. The prominent DSB repair pathway in humans is non-homologous end-joining (NHEJ). In the simplest sense, NHEJ mediates the direct re-ligation of the broken DNA molecule. However, NHEJ is a complex and versatile process that can repair DSBs with a variety of damages and ends via the utilization of a significant number of proteins. In this review we will describe the important factors and mechanisms modulating NHEJ with emphasis given to the versatility of this repair process and the DNA-PK complex.     

The relationships between XPC binding to conformationally diverse DNA adducts and their excision by the human NER system: Is there a correlation?

The first eukaryotic NER factor that recognizes NER substrates is the heterodimeric XPC-RAD23B protein. The currently accepted hypothesis is that this protein recognizes the distortions/destabilization caused by DNA lesions rather than the lesions themselves. The resulting XPC-RAD23B–DNA complexes serve as scaffolds for the recruitment of subsequent NER factors that lead to the excision of the oligonucleotide sequences containing the lesions. Based on several well-known examples of DNA lesions like the UV radiation-induced CPD and 6–4 photodimers, as well as cisplatin-derived intrastrand cross-linked lesions, it is generally believed that the differences in excision activities in human cell extracts is correlated with the binding affinities of XPC-RAD23B to these DNA lesions. However, using electrophoretic mobility shift assays, we have found that XPC-RAD23B binding affinities of certain bulky lesions derived from metabolically activated polycyclic aromatic hydrocarbon compounds such as benzo[a]pyrene and dibenzo[a,l]pyrene, are not directly, or necessarily correlated with NER excision activities observed in cell-free extracts. These findings point to features of XPC-RAD23B–bulky DNA adduct complexes that may involve the formation of NER-productive or unproductive forms of binding that depend on the structural and stereochemical properties of the DNA adducts studied. The pronounced differences in NER cleavage efficiencies observed in cell-free extracts may be due to differences in the successful recruitment of subsequent NER factors by the XPC-RAD23B–DNA adduct complexes, and/or in the verification step. These phenomena appear to depend on the structural and conformational properties of the class of bulky DNA adducts studied.     

LAMMER kinase contributes to genome stability in Ustilago maydis

Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1^Q488*) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1^Q488* rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1^Q488* mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1^Q488* mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis.     

Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair

Transplantation of bone-marrow derived mesenchymal stem cells (MSCs) has potential therapeutic effects on cardiac muscle repair. However, the underlying mechanism remains not completely clarified. Here we show that transplantation of MSCs significantly increased local recruitment of macrophages to facilitate cardiac muscle repair. MSCs-induced recovery of cardiac function and attenuation of fibrosis after injury were all abolished by either impaired macrophage infiltration, or by MSCs depletion after macrophage recruitment. However, angiogenesis seemed to be only affected by depletion of macrophages, but not by depletion of MSCs, suggesting that macrophages are responsible for the augmented angiogenesis after MSCs transplantation, while MSCs do not directly contribute to angiogenesis in the functional cardiac repair. Moreover, high level of transforming growth factor β 1 (TGFβ1) was detected in macrophages and high level of BMP7 was detected in MSCs, suggesting that MSCs not only may recruit macrophages to enhance angiogenesis to promote regeneration, but also may secrete BMP7 to contradict the fibrogenic effect of TGFβ1 by macrophages. Our study thus sheds new insight on the interaction of MSCs and macrophages in a functional cardiac repair triggered by MSCs transplantation.