Eukaryotic DNA damage checkpoint activation in response to double-strand breaks

Double-strand breaks (DSBs) are the most detrimental form of DNA damage. Failure to repair these cytotoxic lesions can result in genome rearrangements conducive to the development of many diseases, including cancer. The DNA damage response (DDR) ensures the rapid detection and repair of DSBs in order to maintain genome integrity. Central to the DDR are the DNA damage checkpoints. When activated by DNA damage, these sophisticated surveillance mechanisms induce transient cell cycle arrests, allowing sufficient time for DNA repair. Since the term “checkpoint” was coined over 20 years ago, our understanding of the molecular mechanisms governing the DNA damage checkpoint has advanced significantly. These pathways are highly conserved from yeast to humans. Thus, significant findings in yeast may be extrapolated to vertebrates, greatly facilitating the molecular dissection of these complex regulatory networks. This review focuses on the cellular response to DSBs in Saccharomyces cerevisiae, providing a comprehensive overview of how these signalling pathways function to orchestrate the cellular response to DNA damage and preserve genome stability in eukaryotic cells.     

Defending the genome from the enemy within: mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline.     

The BLM dissolvasome in DNA replication and repair

RecQ DNA helicases are critical for proper maintenance of genomic stability, and mutations in multiple human RecQ genes are linked with genetic disorders characterized by a predisposition to cancer. RecQ proteins are conserved from prokaryotes to humans and in all cases form higher-order complexes with other proteins to efficiently execute their cellular functions. The focus of this review is a conserved complex that is formed between RecQ helicases and type-I topoisomerases. In humans, this complex is referred to as the BLM dissolvasome or BTR complex, and is comprised of the RecQ helicase BLM, topoisomerase IIIα, and the RMI proteins. The BLM dissolvasome functions to resolve linked DNA intermediates without exchange of genetic material, which is critical in somatic cells. We will review the history of this complex and highlight its roles in DNA replication, recombination, and repair. Additionally, we will review recently established interactions between BLM dissolvasome and a second set of genome maintenance factors (the Fanconi anemia proteins) that appear to allow coordinated genome maintenance efforts between the two systems.     

Chromatin affinity-precipitation using a small metabolic molecule: its application to analysis of <Emphasis Type="Italic">O</Emphasis>-acetyl-ADP-ribose

In the cell, many small endogenous metabolic molecules are involved in distinct cellular functions such as modulation of chromatin structure and regulation of gene expression. O-acetyl-ADP-ribose (AAR) is a small metabolic molecule that is generated during NAD-dependent deacetylation by Sir2. Sir2 regulates gene expression, DNA repair, and genome stability. Here, we developed a novel chromatin affinity-precipitation (ChAP) method to detect the chromatin fragments at which small molecules interact with binding partners. We used this method to demonstrate that AAR associated with heterochromatin. Moreover, we applied the ChAP method to whole genome tiling array chips to compare the association of AAR and Sir2. We found that AAR and Sir2 displayed similar genomic binding patterns. Furthermore, we identified 312 potential association cluster regions of AAR. The ChAP assay may therefore be a generally useful strategy to study the small molecule association with chromosomal regions. Our results further suggest that the small metabolic molecule AAR associates with silent chromatin regions in a Sir2-dependent manner and provide additional support for the role of AAR in assembly of silent chromatin.     

S-phase checkpoint regulations that preserve replication and chromosome integrity upon dNTP depletion

DNA replication stress, an important source of genomic instability, arises upon different types of DNA replication perturbations, including those that stall replication fork progression. Inhibitors of the cellular pool of deoxynucleotide triphosphates (dNTPs) slow down DNA synthesis throughout the genome. Following depletion of dNTPs, the highly conserved replication checkpoint kinase pathway, also known as the S-phase checkpoint, preserves the functionality and structure of stalled DNA replication forks and prevents chromosome fragmentation. The underlying mechanisms involve pathways extrinsic to replication forks, such as those involving regulation of the ribonucleotide reductase activity, the temporal program of origin firing, and cell cycle transitions. In addition, the S-phase checkpoint modulates the function of replisome components to promote replication integrity. This review summarizes the various functions of the replication checkpoint in promoting replication fork stability and genome integrity in the face of replication stress caused by dNTP depletion.     

The inherited susceptibility to cancer

The study of inherited cancer syndromes has led to the identification of over 25 genes directly involved in tumorigenesis. These genes have functions as diverse as signal transduction, cell cycle control, cell-to-cell adhesion, control of apoptosis, DNA repair and the maintenance of genome stability. Most cancer syndromes have a dominant pattern of inheritance, due to germline loss-of-function mutation of a tumour suppressor gene. All the recessively inherited genes have been implicated in the maintenance of genome stability. This review summarises our current understanding of the functions of the major cancer susceptibility genes.     

生物医学研究热点及意义

生物医学研究是目前发展最快、令人振奋的科技领域。本文概述了生物医学的发展趋势，并对生物医学前沿领域的研究热点，如基因组、蛋白质组、细胞信号转导机制、干细胞研究、神经科学研究、生物信息学等进行了分析。对生物技术领域的研究热点，如生物芯片技术、高效基因转移系统技术、组织工程技术和干细胞定向分化技术、基因敲除和敲入技术、克隆技术等进行了综述。提出研究中应该注意针对研究热点选题，注意多学科交叉和集团作战，同时注意知识产权保护。     

Let-7b is a novel regulator of hepatitis C virus replication

The non-coding microRNA (miRNA) is involved in the regulation of hepatitis C virus (HCV) infection and offers an alternative target for developing anti-HCV agent. In this study, we aim to identify novel cellular miRNAs that directly target the HCV genome with anti-HCV therapeutic potential. Bioinformatic analyses were performed to unveil liver-abundant miRNAs with predicted target sequences on HCV genome. Various cell-based systems confirmed that let-7b plays a negative role in HCV expression. In particular, let-7b suppressed HCV replicon activity and down-regulated HCV accumulation leading to reduced infectivity of HCVcc. Mutational analysis identified let-7b binding sites at the coding sequences of NS5B and 5'-UTR of HCV genome that were conserved among various HCV genotypes. We further demonstrated that the underlying mechanism for let-7b-mediated suppression of HCV RNA accumulation was not dependent on inhibition of HCV translation. Let-7b and IFNα-2a also elicited a synergistic inhibitory effect on HCV infection. Together, let-7b represents a novel cellular miRNA that targets the HCV genome and elicits anti-HCV activity. This study thereby sheds new insight into understanding the role of host miRNAs in HCV pathogenesis and to developing a potential anti-HCV therapeutic strategy.     

Metabolic iteration, evolution and cognition in cellular proliferation

A model for cellular proliferation is described according to which proliferation ensues when metabolism evolves towards commitment to DNA synthesis, and inhibition of proliferation occurs when enzymic interactions are iterated within a few metabolic pathways, another limiting factor being the supply of metabolites. The model successfully describes cellular growth and division as a 'cognitive process' based on interaction within enzymic elements and the genome, and affords an explanation in these terms of some empirical phenomena which have previously been understood only as isolated observations.     

Chromosome instability and deregulated proliferation: an unavoidable duo

The concept that aneuploidy is a characteristic of malignant cells has long been known; however, the idea that aneuploidy is an active contributor to tumorigenesis, as opposed to being an associated phenotype, is more recent in its evolution. At the same time, we are seeing the emergence of novel roles for tumor suppressor genes and oncogenes in genome stability. These include the adenomatous polyposis coli gene (APC), p53, the retinoblastoma susceptibility gene (RB1), and Ras. Originally, many of these genes were thought to be tumor suppressive or oncogenic solely because of their role in proliferative control. Because of the frequency with which they are disrupted in cancer, chromosome instability caused by their dysfunction may be more central to tumorigenesis than previously thought. Therefore, this review will highlight how the proper function of cell cycle regulatory genes contributes to the maintenance of genome stability, and how their mutation in cancer obligatorily connects proliferation and chromosome instability.     

Murine type C endogenous virus expression in murine 129 teratocarcinoma cell line]

Murine type C viral information is detectable in the cellular genome of both differentiated and undifferentiated cell lines derived from 129 Mouse teratocarcinoma. Cytoplasmic RNA expression which is negligible in differentiated cells, is significantly higher in multipotential undifferentiated cells. Furthermore it was observed that in vitro differentiation of multipotent cells leads to a decrease of this expression.     

Developmental control of heat shock and chaperone gene expression Hsp70 genes and heat shock factors during preimplantation phase of mouse development

Heat shock genes are found in all organisms, and synthesis of heat shock proteins is induced by various stressors in nearly all the cells forming these organisms. However, a particular situation is noticed for hsp70 genes in mouse embryos at the beginning of their development. First, spontaneous expression of hsp70 is observed at the onset of zygotic genome activity. Second, inducible expression is delayed until morula or early blastocyst stages. A better understanding of both these points depends on a more careful analysis of hsp70 expression in relation to their major regulators, the heat shock factors. In this review, we will see how the development of the preimplanta tion embryo highlights the complexity of heat shock gene regulation involving trans-cis interactions and the cellular and nuclear environment.     

Endonuclease V: an unusual enzyme for repair of DNA deamination

Endonuclease V (endo V) was first discovered as the fifth endonuclease in Escherichia coli in 1977 and later rediscovered as a deoxyinosine 3′ endonuclease. Decades of biochemical and genetic investigations have accumulated rich information on its role as a DNA repair enzyme for the removal of deaminated bases. Structural and biochemical analyses have offered invaluable insights on its recognition capacity, catalytic mechanism, and multitude of enzymatic activities. The roles of endo V in genome maintenance have been validated in both prokaryotic and eukaryotic organisms. The ubiquitous nature of endo V in the three domains of life: Bacteria, Archaea, and Eukaryotes, indicates its existence in the early evolutionary stage of cellular life. The application of endo V in mutation detection and DNA manipulation underscores its value beyond cellular DNA repair. This review is intended to provide a comprehensive account of the historic aspects, biochemical, structural biological, genetic and biotechnological studies of this unusual DNA repair enzyme.     

Regulation of the cell cycle following DNA damage in normal and Ataxia telangiectasia cells

A proportion of the population is exposed to acute doses of ionizing radiation through medical treatment or occupational accidents, with little knowledge of the immedate effects. At the cellular level, ionizing radiation leads to the activation of a genetic program which enables the cell to increase its chances of survival and to minimize detrimental manifestations of radiation damage. Cytotoxic stress due to ionizing radiation causes genetic instability, alterations in the cell cycle, apoptosis, or necrosis. Alterations in the G1, S and G2 phases of the cell cycle coincide with improved survival and genome stability. The main cellular factors which are activated by DNA damage and interfere with the cell cycle controls are: p53, delaying the transition through the G1-S boundary; p21^WAF1/CIPI, preventing the entrance into S-phase; proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), blocking DNA replication; and the p53 variant protein p53as together with the retinoblastoma protein (Rb), with less defined functions during the G2 phase of the cell cycle. By comparing a variety of radioresistant cell lines derived from radiosensitive ataxia talangiectasia cells with the parental cells, some essential mechanisms that allow cells to gain radioresistance have been identified. The results so far emphasise the importance of an adequate delay in the transition from G2 to M and the inhibition of DNA replication in the regulation of the cell cycle after exposure to ionizing radiation.     

Functional interplay between Parp-1 and SirT1 in genome integrity and chromatin-based processes

Poly(ADP-ribose) polymerase-1 (Parp-1) and the protein deacetylase SirT1 are two of the most effective NAD⁺-consuming enzymes in the cell with key functions in genome integrity and chromatin-based pathways. Here, we examined the in vivo crosstalk between both proteins. We observed that the double disruption of both genes in mice tends to increase late post-natal lethality before weaning consistent with important roles of both proteins in genome integrity during mouse development. We identified increased spontaneous telomeric abnormalities associated with decreased cell growth in the absence of either SirT1 or SirT1 and Parp-1 in mouse cells. In contrast, the additional disruption of Parp-1 rescued the abnormal pericentric heterochromatin, the nucleolar disorganization and the mitotic defects observed in SirT1-deficient cells. Together, these findings are in favor of key functions of both proteins in cellular response to DNA damage and in the modulation of histone modifications associated with constitutive heterochromatin integrity.     

Recent insights into the biology and biomedical applications of Flock House virus

Flock House virus (FHV) is a nonenveloped, icosahedral insect virus whose genome consists of two molecules of single-stranded, positive-sense RNA. FHV is a highly tractable system for studies on a variety of basic aspects of RNA virology. In this review, recent studies on the replication of FHV genomic and subgenomic RNA are discussed, including a landmark study on the ultrastructure and molecular organization of FHV replication complexes. In addition, we show how research on FHV B2, a potent suppressor of RNA silencing, resulted in significant insights into antiviral immunity in insects. We also explain how the specific packaging of the bipartite genome of this virus is not only controlled by specific RNA-protein interactions but also by coupling between RNA replication and genome recognition. Finally, applications for FHV as an epitopepresenting system are described with particular reference to its recent use for the development of a novel anthrax antitoxin and vaccine.     

The molecular role of the Rothmund-Thomson-, RAPADILINO- and Baller-Gerold-gene product, RECQL4: recent progress

The RecQ family of DNA helicases is highly conserved throughout evolution and plays an important role in the maintenance of genomic stability in all organisms. Mutations in three of the five known family members in humans, BLM, WRN and RECQL4, give rise to disorders that are characterized by predisposition to cancer and premature aging, emphasizing the importance of studying the RecQ proteins and their cellular activities. Interestingly, three autosomal recessive disorders have been associated with mutations in the RECQL4 gene: Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes, thus making RECQL4 unique within the RecQ family of DNA helicases. To date, however, the molecular function of RECQL4 and the possible cellular pathways in which it is involved remain poorly understood. Here, we present an overview of recent findings in connection with RECQL4 and try to highlight different directions the field could head, helping to clarify the role of RECQL4 in preventing tumorigenesis and maintenance of genome integrity in humans. Received 31 October 2006; received after revision 4 January 2007; accepted 5 February 2007