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.
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"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: 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.
"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 . "
"The genomic DNA of cells is constantly challenged by exogenous and endogenous DNA-damaging events (Hakem, 2008). Double-stranded DNA (dsDNA) breaks can occur as often as 50 times per cell cycle in human cells (Vilenchik and Knudson, 2003). "