Maintaining integrity

Research on genome stability and integrity now extends far beyond the biochemistry of DNA repair to encompass signal transduction pathways that span numerous aspects of cellular life. Derailed genomic integrity pathways can result in debilitating genetic disorders, premature ageing, predisposition to cancer and degenerative conditions. Current progress in this rapidly expanding field was the subject of an EMBO workshop, Maintenance of Genomic Integrity, that took place in June 2004 in Galway, Ireland. Top     

Maintaining data integrity in microarray data management

Gene expression microarrays are a relatively new technology, dating back just a few years, yet they have already become a very widely used tool in biology, and have evolved to a wide range of applications well beyond their original design intent. However, while the use of microarrays has expanded, and the issues of performance optimization have been intensively studied, the fundamental issue of data integrity management has largely been ignored. Now that performance has improved so greatly, the shortcomings of data integrity control methods constitute a greater percent of the stumbling blocks for investigators. Microarray data are cumbersome, and the rule up to this point has mostly been one of hands-on transformations, leading to human errors which often have dramatic consequences. We show in this review that the time lost on such mistakes is enormous and dramatically affects results; therefore, mistakes should be mitigated in any way possible. We outline the scope of the data integrity issue, to survey some of the most common and dangerous data transformations, and their shortcomings. To illustrate, we review some case studies. We then look at the work done by the research community on this issue (which admittedly is meager up to this point). Some data integrity issues are always going to be difficult, while others will become easier-one of our goals is to expedite the use of integrity control methods. Finally, we present some preliminary guidelines and some specific approaches that we believe should be the focus of future research.     

Guarding genome integrity in stem cells

Comment on: Mantel C, et al. Cell Cycle 2010; 9:In press.     

Maintaining replication fork integrity in UV-irradiated Escherichia coli cells

In dividing cells, the stalling of replication fork complexes by impediments to DNA unwinding or by template imperfections that block synthesis by the polymerase subunits is a serious threat to genomic integrity and cell viability. What happens to stalled forks depends on the nature of the offending obstacle. In UV-irradiated Escherichia coli cells DNA synthesis is delayed for a considerable period, during which forks undergo extensive processing before replication can resume. Thus, restart depends on factors needed to load the replicative helicase, indicating that the replisome may have dissociated. It also requires the RecFOR proteins, which are known to load RecA recombinase on single-stranded DNA, implying that template strands are exposed. To gain a further understanding of how UV irradiation affects replication and how replication resumes after a block, we used fluorescence microscopy and BrdU or radioisotope labelling to examine chromosome replication and cell cycle progression. Our studies confirm that RecFOR promote efficient reactivation of stalled forks and demonstrate that they are also needed for productive replication initiated at the origin, or triggered elsewhere by damage to the DNA. Although delayed, all modes of replication do recover in the absence of these proteins, but nascent DNA strands are degraded more extensively by RecJ exonuclease. However, these strands are also degraded in the presence of RecFOR when restart is blocked by other means, indicating that RecA loading is not sufficient to stabilise and protect the fork. This is consistent with the idea that RecA actively promotes restart. Thus, in contrast to eukaryotic cells, there may be no factor in bacterial cells acting specifically to stabilise stalled forks. Instead, nascent strands may be protected by the simple expedient of promoting restart. We also report that the efficiency of fork reactivation is not affected in polB mutants.     

Cohesin: A guardian of genome integrity

Ability to reproduce is one of the hallmark features of all life forms by which new organisms are produced from their progenitors. During this process each cell duplicates its genome and passes a copy of its genome to the daughter cells along with the cellular matrix. Unlike bacteria, in eukaryotes there is a definite time gap between when the genome is duplicated and when it is physically separated. Therefore, for precise halving of the duplicated genome into two, it is required that each pair of duplicated chromosomes, termed sister chromatids, should be paired together in a binary fashion from the moment they are generated. This pairing function between the duplicated genome is primarily provided by a multimeric protein complex, called cohesin. Thus, genome integrity largely depends on cohesin as it ensures faithful chromosome segregation by holding the sister chromatids glued together from S phase to anaphase. In this review, we have discussed the life cycle of cohesin during both mitotic and meiotic cell divisions including the structure and architecture of cohesin complex, relevance of cohesin associated proteins, mechanism of cohesin loading onto the chromatin, cohesion establishment and the mechanism of cohesin disassembly during anaphase to separate the sister chromatids. We have also focused on the role of posttranslational modifications in cohesin biology. For better understanding of the complexity of the cohesin regulatory network to the readers, we have presented an interactome profiling of cohesin core subunits in budding yeast during mitosis and meiosis.     

Maintaining the integrity of human immunodeficiency virus sequence databases.

Human immunodeficiency virus type 1 (HIV-1) sequences are accumulating in the literature at a rapid pace. For this ever-expanding resource to be maximally useful, it is critical that researchers strive to maintain a high level of quality assurance, both in experimental design and conduct and in analyses. Here we present detailed analyses of problematic sets of HIV-1 sequences in the database that include sequence anomalies suggestive of mislabeling or sample contamination problems. These data are examined in the context of currently available HIV-1 sequence information to provide an example of how to identify potentially flawed data. Indicators of potential problems with sequences are (i) sequences that are nearly identical that are supposed to be derived from unlinked individuals and that are markedly distinct from other sequences from the putative source or (ii) sequences that are nearly identical to those of laboratory strains. We provide an outline of methods that researchers can use to perform preliminary laboratory and computational analyses that could help identify problematic data and thus help ensure the integrity of sequence databases.     

Maintaining corneal integrity how the "window" stays clear

The anterior surface of the eye is composed of the cornea, conjunctiva, and the zone between the two called the limbus. The cornea must maintain optical clarity to retain good vision. However, the ocular surface is vulnerable to trauma, microbial infection, and exposure to environmental toxins. This places the cornea, especially, at risk for disruptions of the epithelial barrier and subsequent immunopathological events. Cell-cell and cell-matrix attachment junctions incorporating adhesion molecules ensure that the epithelial barrier remains intact. Protein components of the basement membrane, including laminins, are vital to the adhesion of corneal epithelial cells to the underlying stroma and function to enhance the strength of the bond between epithelium and connective tissue. Epithelial cells also play an early and crucial role in the initiation of ocular surface responses should a potentially antigenic molecule enter into deeper corneal tissues. For example, epithelial cells may produce and release cytokines such as interleukin-1 (IL-1). The delicate balance between the matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are central to mechanisms regulating dissolution of the extracellular matrix that may be a consequence of infection or wound healing. Adhesion molecules, cytokines and chemokines, and MMPs and TIMPs thus participate in the corneal response to immunologic challenge or wounding. They may also be involved in corneal pathologies associated with genetic diseases, diabetes, and vitamin A deficiency. In addition these molecules are components of cellular pathways underlying the clinical complications often observed with contact lens wear and refractive surgeries used to improve visual acuity.     

ATR: an essential regulator of genome integrity

Genome maintenance is a constant concern for cells, and a coordinated response to DNA damage is required to maintain cellular viability and prevent disease. The ataxia-telangiectasia mutated (ATM) and ATM and RAD3-related (ATR) protein kinases act as master regulators of the DNA-damage response by signalling to control cell-cycle transitions, DNA replication, DNA repair and apoptosis. Recent studies have provided new insights into the mechanisms that control ATR activation, have helped to explain the overlapping but non-redundant activities of ATR and ATM in DNA-damage signalling, and have clarified the crucial functions of ATR in maintaining genome integrity.     

Chromatin remodeling and the maintenance of genome integrity

DNA damage of any type is threatening for a cell. If lesions are left unrepaired, genomic instability can arise, faithful transmission of genetic information is greatly compromised eventually leading the cell to undergo apoptosis or carcinogenesis. In order to access/detect and repair these damages, repair factors must circumvent the natural repressive barrier of chromatin. This review will present recent progress showing the intricate link between chromatin, its remodeling and the DNA repair process. Several studies demonstrated that one of the first events following specific types of DNA damage is the phosphorylation of histone H2A. This mark or the damage itself are responsible for the association of chromatin-modifying complexes near damaged DNA. These complexes are able to change the chromatin structure around the wounded DNA in order to allow the repair machinery to gain access and repair the lesion. Chromatin modifiers include ATP-dependent remodelers such as SWI/SNF and Rad54 as well as histone acetyltransferases (HATs) like SAGA/NuA4-related complexes and p300/CBP, which have been shown to facilitate DNA accessibility and repair in different pathways leading to the maintenance of genome integrity.     

A MyoD-dependent differentiation checkpoint: ensuring genome integrity

In a recent paper, the concept of a genotoxic stress-induced differentiation checkpoint has been proposed. The suggested function of this checkpoint is to preserve the integrity of the genome in terminally differentiated cells.     

Caretaker tumour suppressor genes that defend genome integrity

Cancers arise as a result of genetic changes that impact upon cell proliferation through promoting cell division and/or inhibiting cell death. Tumour suppressor (TS) genes are the targets for many of these genetic changes. In general, both alleles of TS genes must be disrupted to observe a phenotypic effect. Broadly speaking, there are two types of TS gene: 'gatekeepers' and 'caretakers'. In contrast to gatekeepers, caretaker genes do not directly regulate proliferation, but act to prevent genomic instability. Thus, mutation of caretaker genes leads to accelerated conversion of a normal cell to a neoplastic cell. Many caretaker genes are required for the maintenance of genome integrity. This review focuses on those caretaker genes that play a role, directly or indirectly, in the repair of DNA strand breaks by the homologous recombination pathway, and that are associated with cancer-prone clinical syndromes, in particular ataxia telangiectasia, hereditary breast cancer, Bloom's syndrome and Werner's syndrome.     

piRNAs, transposon silencing, and germline genome integrity

Integrity of the germline genome is essential for the production of viable gametes and successful reproduction. In mammals, the generation of gametes involves extensive epigenetic changes (DNA methylation and histone modification) in conjunction with changes in chromosome structure to ensure flawless progression through meiotic recombination and packaging of the genome into mature gametes. Although epigenetic reprogramming is essential for mammalian reproduction, reprogramming also provides a permissive window for exploitation by transposable elements (TEs), autonomously replicating endogenous elements. Expression and propagation of TEs during the reprogramming period can result in insertional mutagenesis that compromises genome integrity leading to reproductive problems and sporadic inherited diseases in offspring. Recent work has identified the germ cell associated PIWI Interacting RNA (piRNA) pathway in conjunction with the DNA methylation and histone modification machinery in silencing TEs. In this review we will highlight these recent advances in piRNA mediated regulation of TEs in the mouse germline, as well as mention the repercussions of failure to properly regulate TEs.     

Proteolytic control of genome integrity at the replication fork

Faithful duplication of the genome is critical for the survival of an organism and prevention of malignant transformation. Accurate replication of a large amount of genetic information in a timely manner is one of the most challenging cellular processes and is often perturbed by intrinsic and extrinsic barriers to DNA replication fork progression, a phenomenon referred to as DNA replication stress. Elevated DNA replication stress is a primary source of genomic instability and one of the key hallmarks of cancer. Therefore, targeting DNA replication stress is an emerging concept for cancer therapy. The replication machinery associated with PCNA and other regulatory factors coordinates the synthesis and repair of DNA strands at the replication fork. The dynamic interaction of replication protein complexes with DNA is essential for sensing and responding to various signaling events relevant to DNA replication and damage. Thus, the disruption of the spatiotemporal regulation of protein homeostasis at the replication fork impairs genome integrity, which often involves the deregulation of ubiquitin-mediated proteolytic signaling. Notably, emerging evidence has highlighted the role of the AAA+ATPase VCP/p97 in extracting ubiquitinated protein substrates from the chromatin and facilitating the turnover of genome surveillance factors during DNA replication and repair. Here, we review recent advances in our understanding of chromatin-associated degradation pathways at the replication fork and the implication of these findings for cancer therapy.     

Nuclear pore complexes in the maintenance of genome integrity

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