Implication of the VRK1 chromatin kinase in the signaling responses to DNA damage: a therapeutic target?

DNA damage causes a local distortion of chromatin that triggers the sequential processes that participate in specific DNA repair mechanisms. This initiation of the repair response requires the involvement of a protein whose activity can be regulated by histones. Kinases are candidates to regulate and coordinate the connection between a locally altered chromatin and the response initiating signals that lead to identification of the type of lesion and the sequential steps required in specific DNA damage responses (DDR). This initiating kinase must be located in chromatin, and be activated independently of the type of DNA damage. We review the contribution of the Ser-Thr vaccinia-related kinase 1 (VRK1) chromatin kinase as a new player in the signaling of DNA damage responses, at chromatin and cellular levels, and its potential as a new therapeutic target in oncology. VRK1 is involved in the regulation of histone modifications, such as histone phosphorylation and acetylation, and in the formation of γH2AX, NBS1 and 53BP1 foci induced in DDR. Induction of DNA damage by chemotherapy or radiation is a mainstay of cancer treatment. Therefore, novel treatments can be targeted to proteins implicated in the regulation of DDR, rather than by directly causing DNA damage.     

Functions of disordered regions in mammalian early base excision repair proteins

Reactive oxygen species, generated endogenously and induced as a toxic response, produce several dozen oxidized or modified bases and/or single-strand breaks in mammalian and other genomes. These lesions are predominantly repaired via the conserved base excision repair (BER) pathway. BER is initiated with excision of oxidized or modified bases by DNA glycosylases leading to formation of abasic (AP) site or strand break at the lesion site. Structural analysis by experimental and modeling approaches shows the presence of a disordered segment commonly localized at the N- or C-terminus as a characteristic signature of mammalian DNA glycosylases which is absent in their bacterial prototypes. Recent studies on unstructured regions in DNA metabolizing proteins have indicated their essential role in interaction with other proteins and target DNA recognition. In this review, we have discussed the unique presence of disordered segments in human DNA glycosylases, and AP endonuclease involved in the processing of glycosylase products, and their critical role in regulating repair functions. These disordered segments also include sites for posttranslational modifications and nuclear localization signal. The teleological basis for their structural flexibility is discussed.     

ATM protein kinase: the linchpin of cellular defenses to stress

ATM is the most significant molecule involved in monitoring the genomic integrity of the cell. Any damage done to DNA relentlessly challenges the cellular machinery involved in recognition, processing and repair of these insults. ATM kinase is activated early to detect and signal lesions in DNA, arrest the cell cycle, establish DNA repair signaling and faithfully restore the damaged chromatin. ATM activation plays an important role as a barrier to tumorigenesis, metabolic syndrome and neurodegeneration. Therefore, studies of ATM-dependent DNA damage signaling pathways hold promise for treatment of a variety of debilitating diseases through the development of new therapeutics capable of modulating cellular responses to stress. In this review, we have tried to untangle the complex web of ATM signaling pathways with the purpose of pinpointing multiple roles of ATM underlying the complex phenotypes observed in AT patients.     

The dual role model for p53 in maintaining genomic integrity

The tumour suppressor p53 is a potent mediator of cellular responses against genotoxic insults. In this review we describe the multiple functions of p53 in response to DNA damage, with an emphasis on p53's role in DNA repair. We summarize data demonstrating that p53 actively participates in various processes of DNA repair and DNA recombination via its ability to interact with components of the repair and recombination machinery, and by its various biochemical activities. An important aspect in evaluating p53 functions is provided by the finding that the core domain of p53 harbours two mutually exclusive biochemical activities, sequence-specific DNA binding required for its transactivation function, and 3'-5' exonuclease activity, possibly involved in aspects of DNA repair. Based on the finding that modifications of p53 which lead to activation of its sequence-specific DNA-binding activity result in inactivation of its 3'-5' exonuclease activity, we propose that p53 exerts its functions as a 'guardian of the genome' at various levels: in its noninduced state, p53 should not be regarded as a 'dead' protein but, for example, via its exonuclease activity might be actively involved in prevention and repair of endogenous DNA damage. Upon induction through exogenous DNA damage, p53 will exert its well-documented functions as a superior response element in various types of cellular stress. This dual role model for p53 in maintaining genomic integrity significantly enhances p53's possibilities as a guardian of the genome.     

Roles of the DNA mismatch repair and nucleotide excision repair proteins during meiosis

Numerous proteins are involved in the nucleotide excision repair (NER) and DNA mismatch repair (MMR) pathways. The function and specificity of these proteins during the mitotic cell cycle has been actively investigated, in large part due to the involvement of these systems in human diseases. In contrast, comparatively little is known about their functioning during meiosis. At least three repair pathways operate during meiosis in the yeast Saccharomyces cerevisiae to repair mismatches that occur as a consequence of heteroduplex formation in recombination. The first pathway is similar to the one acting during postreplicative mismatch repair in mitotically dividing cells, while two pathways are responsible for the repair of large loops during meiosis, using proteins from MMR and NER systems. Some MMR proteins also help prevent recombination between diverged sequences during meiosis, and act late in recombination to affect the resolution of crossovers. This review will discuss the current status of DNA mismatch repair and nucleotide excision repair proteins during meiosis, especially in the yeast S. cerevisiae. Received 21 September 1998; received after revision 23 November 1998; accepted 23 November 1998     

Theearly andlate processing of lysosomal enzymes: Proteolysis and compartmentation

Lysosomal enzymes are subjected to a number of modifications including carbohydrate restructuring and proteolytic maturation. Some of these reactions support lysosomal targeting, others are necessary for activation or keeping the enzyme inactive before being segregated, while still others may be adventitious. The non-segregated fraction of the enzyme is secreted and can be isolated from the medium. It is considered that the secreted lysosomal enzymes fulfill certain physiological and pathophysiological roles. By comparing the secreted and the intracellular enzymes it is possible to distinguish between the reactions that occur before and after the segregation. In this review the reactions that may influence the segregation are referred to as the early processing and those characteristic for the enzymes isolated from lysosomal compartments as the late processing. The early processing is characterized mainly by modifications of carbohydrate side chains. In the late processing, proteolytic fragmentation represents the most conspicuous changes. The review focuses on the compartmentation of the reactions and the proteolytic fragmentation of lysosomal enzyme precursors. While a plethora of proteolytic reactions are involved, our knowledge of the proteinases responsible for the particular maturation reactions remains very limited. The review points also to work with cells from patients affected with lysosomal storage disorders, which contributed to our understanding of the lysosomal apparatus.     

Silencing of RB1 but not of RB2/P130 induces cellular senescence and impairs the differentiation potential of human mesenchymal stem cells

Stem cell senescence is considered deleterious because it may impair tissue renewal and function. On the other hand, senescence may arrest the uncontrolled growth of transformed stem cells and protect organisms from cancer. This double function of senescence is strictly linked to the activity of genes that the control cell cycle such as the retinoblastoma proteins RB1, RB2/P130, and P107. We took advantage of the RNA interference technique to analyze the role of these proteins in the biology of mesenchymal stem cells (MSC). Cells lacking RB1 were prone to DNA damage. They showed elevated levels of p53 and p21^cip1 and increased regulation of RB2/P130 and P107 expression. These cells gradually adopted a senescent phenotype with impairment of self-renewal properties. No significant modification of cell growth was observed as it occurs in other cell types or systems. In cells with silenced RB2/P130, we detected a reduction of DNA damage along with a higher proliferation rate, an increase in clonogenic ability, and the diminution of apoptosis and senescence. Cells with silenced RB2/P130 were cultivated for extended periods of time without adopting a transformed phenotype. Of note, acute lowering of P107 did not induce relevant changes in the in vitro behavior of MSC. We also analyzed cell commitment and the osteo-chondro-adipogenic differentiation process of clones derived by MSC cultures. In all clones obtained from cells with silenced retinoblastoma genes, we observed a reduction in the ability to differentiate compared with the control clones. In summary, our data show evidence that the silencing of the expression of RB1 or RB2/P130 is not compensated by other gene family members, and this profoundly affects MSC functions.     

The early and late processing of lysosomal enzymes: proteolysis and compartmentation.

Lysosomal enzymes are subjected to a number of modifications including carbohydrate restructuring and proteolytic maturation. Some of these reactions support lysosomal targeting, others are necessary for activation or keeping the enzyme inactive before being segregated, while still others may be adventitious. The non-segregated fraction of the enzyme is secreted and can be isolated from the medium. It is considered that the secreted lysosomal enzymes fulfill certain physiological and pathophysiological roles. By comparing the secreted and the intracellular enzymes it is possible to distinguish between the reactions that occur before and after the segregation. In this review the reactions that may influence the segregation are referred to as the early processing and those characteristic for the enzymes isolated from lysosomal compartments as the late processing. The early processing is characterized mainly by modifications of carbohydrate side chains. In the late processing, proteolytic fragmentation represents the most conspicuous changes. The review focuses on the compartmentation of the reactions and the proteolytic fragmentation of lysosomal enzyme precursors. While a plethora of proteolytic reactions are involved, our knowledge of the proteinases responsible for the particular maturation reactions remains very limited. The review points also to work with cells from patients affected with lysosomal storage disorders, which contributed to our understanding of the lysosomal apparatus.