DNA-damage repair; the good, the bad, and the ugly. EMBO J
Organisms have developed several DNA-repair pathways as well as DNA-damage checkpoints to cope with the frequent challenge of endogenous and exogenous DNA insults. In the absence or impairment of such repair or checkpoint mechanisms, the genomic integrity of the organism is often compromised. This review will focus on the functional consequences of impaired DNA-repair pathways. Although each pathway is addressed individually, it is essential to note that cross talk exists between repair pathways, and that there are instances in which a DNA-repair protein is involved in more than one pathway. It is also important to integrate DNA-repair process with DNA-damage checkpoints and cell survival, to gain a better understanding of the consequences of compromised DNA repair at both cellular and organismic levels. Functional consequences associated with impaired DNA repair include embryonic lethality, shortened life span, rapid ageing, impaired growth, and a variety of syndromes, including a pronounced manifestation of cancer.
[Show abstract] [Hide abstract] ABSTRACT: Emergence of drug-resistant Plasmodium falciparum has created an urgent need for new drug targets. DNA polymerase δ is an essential enzyme required for chromosomal DNA replication and repair, and therefore may be a potential target for anti-malarial drug development. However, little is known of the characteristics and function of this P. falciparum enzyme. The coding sequences of DNA polymerase δ catalytic subunit (PfPolδ-cat), DNA polymerase δ small subunit (PfPolδS) and proliferating cell nuclear antigen (PfPCNA) from chloroquine- and pyrimethamine-resistant P. falciparum strain K1 were amplified, cloned into an expression vector and expressed in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE and identified by LC–MS/MS. PfPolδ-cat was biochemically characterized. The roles of PfPolδS and PfPCNA in PfPolδ-cat function were investigated. In addition, inhibitory effects of 11 compounds were tested on PfPolδ-cat activity and on in vitro parasite growth using SYBR Green I assay. The purified recombinant protein PfPolδ-cat, PfPolδS and PfPCNA showed on SDS-PAGE the expected size of 143, 57 and 34 kDa, respectively. Predicted amino acid sequence of the PfPolδ-cat and PfPolδS had 59.2 and 24.7 % similarity respectively to that of the human counterpart. The PfPolδ-cat possessed both DNA polymerase and 3′–5′ exonuclease activities. It used both Mg 2+ and Mn 2+ as cofactors and was inhibited by high KCl salt (>200 mM). PfPolδS stimulated PfPolδ-cat activity threefolds and up to fourfolds when PfPCNA was included in the assay. Only two compounds were potent inhibitors of PfPolδ-cat, namely, butylphenyl-dGTP (BuPdGTP; IC 50 of 38 µM) and 7-acetoxypentyl-(3, 4 dichlorobenzyl) guanine (7-acetoxypentyl-DCBG; IC 50 of 55 µM). The latter compound showed higher inhibition on parasite growth (IC 50 of 4.1 µM). Recombinant PfPolδ-cat, PfPolδS and PfPCNA were successfully expressed and purified. PfPolS and PfPCNA increased DNA polymerase activity of PfPolδ-cat. The high sensitivity of PfPolδ to BuPdGTP can be used to differentiate parasite enzyme from mammalian and human counterparts. Interestingly, 7-acetoxypentyl-DCBG showed inhibitory effects on both enzyme activity and parasite growth. Thus, 7-acetoxypentyl-DCBG is a potential candidate for future development of a new class of anti-malarial agents targeting parasite replicative DNA polymerase.
- "In addition to its function in DNA replication, Pol δ plays a role in DNA repair and recombination . In base excision repair (BER), one of DNA repair mechanisms of single-stranded DNA damage, Pol δ is involved in the long-path pathway, whereas Pol β plays a role in the short-path pathway . Interestingly, the long-patch BER is predominate in P. falciparum while short-path BER is mainly found in humans . "
[Show abstract] [Hide abstract] ABSTRACT: Vascular aging plays a central role in health problems and mortality in older people. Apart from the impact of several classical cardiovascular risk factors on the vasculature, chronological aging remains the single most important determinant of cardiovascular problems. The causative mechanisms by which chronological aging mediates its impact, independently from classical risk factors, remain to be elucidated. In recent years evidence has accumulated that unrepaired DNA damage may play an important role. Observations in animal models and in humans indicate that under conditions during which DNA damage accumulates in an accelerated rate, functional decline of the vasculature takes place in a similar but more rapid or more exaggerated way than occurs in the absence of such conditions. Also epidemiological studies suggest a relationship between DNA maintenance and age-related cardiovascular disease. Accordingly, mouse models of defective DNA repair are means to study the mechanisms involved in biological aging of the vasculature. We here review the evidence of the role of DNA damage in vascular aging, and present mechanisms by which genomic instability interferes with regulation of the vascular tone. In addition, we present potential remedies against vascular aging induced by genomic instability. Central to this review is the role of diverse types of DNA damage (telomeric, non-telomeric and mitochondrial), of cellular changes (apoptosis, senescence, autophagy), mediators of senescence and cell growth (plasminogen activator inhibitor-1 (PAI-1), cyclin-dependent kinase inhibitors, senescence-associated secretory phenotype (SASP)/senescence-messaging secretome (SMS), insulin and insulin-like growth factor 1 (IGF-1) signaling), the adenosine monophosphate-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR)-nuclear factor kappa B (NFκB) axis, reactive oxygen species (ROS) vs. endothelial nitric oxide synthase (eNOS)-cyclic guanosine monophosphate (cGMP) signaling, phosphodiesterase (PDE) 1 and 5, transcription factor NF-E2-related factor-2 (Nrf2), and diet restriction.
- "Each repair system is responsible for a specific subset of lesions, although partial overlap can occur depending on the type of DNA lesion that needs to be repaired. At least six DNA repair pathways can be listed in mammalian cells: (1) the direct reversal pathway, which executes the direct reversal of chemical modifications of nucleotides; (2) mismatch repair (MMR), which repairs base pair mismatches; (3) base excision repair (BER), repairing mainly oxidized and alkylation lesions in the nucleus and mitochondria, as well as single-strand breaks; (4) nucleotide excision repair (NER), to correct transcription-disturbing bulky adducts; (5) homologous recombination (HR); and (6) non-homologous end joining (NHEJ), which correct single-and double-strand breaks [10,13]. Telomere maintenance requires further specialized proteins . "
[Show abstract] [Hide abstract] ABSTRACT: The lymphocyte cytokinesis-block micronucleus (CBMN) assay has been applied in hundreds of in vivo biomonitoring studies of humans exposed either environmentally or occupationally to genotoxic chemicals. However, there is an emerging need to re-evaluate the use of MN and other biomarkers within the lymphocyte CBMN cytome assay as quantitative indicators of exposure to main classes of chemical genotoxins.
- "Efficient DNA repair machinery that removes arising DNA damage, comprising several distinct pathways, ensures effectively genomic integrity. Alterations in the DNA repair increase the vulnerability of the cells, resulting in an accumulation of mutations in the genome, which may ultimately result in tumorigenesis . DNA repair is closely associated with fundamental cellular processes: a) DNA replication -its deregulation occurs through replication-blocking DNA lesions, the activation of certain oncogenes, loss of function of certain tumour suppressors, and promoting replication stress that triggers double-strand breaks (DSB) formation and leads consecutively to unscheduled recombination events and chromosomal rearrangements; b) chromosomal segregation -defects in chromatid cohesion, spindle formation or mitotic checkpoints may lead to aberrant chromosomal segregation; c) telomere maintenance -recognition and signalling of DNA damage is a prerequisite for the induction of subsequent cellular responses such as increased repair, cell cycle arrest and apoptosis . "
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