Peptides containing the PCNA interacting motif APIM bind to the ß-clamp and inhibit bacterial growth and mutagenesis.

In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, ß-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The ß-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the ß-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the ß-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial ß-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents.

AP endonuclease independent repair of abasic sites in Schizosaccharomyces pombe.

Abasic (AP) sites are formed spontaneously and are inevitably intermediates during base excision repair of DNA base damages. AP sites are both mutagenic and cytotoxic and key enzymes for their removal are AP endonucleases. However, AP endonuclease independent repair initiated by DNA glycosylases performing ß,δ-elimination cleavage of the AP sites has been described in mammalian cells. Here, we describe another AP endonuclease independent repair pathway for removal of AP sites in Schizosaccharomyces pombe that is initiated by a bifunctional DNA glycosylase, Nth1 and followed by cleavage of the baseless sugar residue by tyrosyl phosphodiesterase Tdp1. We propose that repair is completed by the action of a polynucleotide kinase, a DNA polymerase and finally a DNA ligase to seal the gap. A fission yeast double mutant of the major AP endonuclease Apn2 and Tdp1 shows synergistic increase in MMS sensitivity, substantiating that Apn2 and Tdp1 process the same substrate. These results add new knowledge to the complex cellular response to AP sites, which could be exploited in chemotherapy where synthetic lethality is a key strategy of treatment.

Up-regulation of myocardial DNA base excision repair activities in experimental heart failure.

Base excision repair (BER) is the major pathway to counteract the genotoxic effect of endogenous DNA damaging agents. The present study investigated the enzymatic activities and gene transcription of DNA glycosylases initiating BER in an experimental heart failure (HF) model. Rats were subjected to myocardial infarction or sham-operated. Twenty-eight days after surgical intervention cell-free protein extracts, total RNA and genomic DNA were isolated to analyze DNA glycosylase and AP-endonuclease activities, transcript levels of DNA glycosylases and accumulation of oxidative DNA damage. The capacity to remove major oxidation products (e.g., formamidopyrimidine and 5-hydroxycytosine) was significantly increased in the border zone of infarcted area, while the capacity to remove the highly mutagenic 8-oxoguanine residue was enhanced both in non-infarcted and infarcted areas of left ventricle (LV). DNA glycosylase activities towards 3-methyladenine and uracil were up-regulated in infarcted and non-infarcted areas of LV, indicating that generation of alkylated and deaminated base lesions on DNA increase during HF. Finally, we found no difference in accumulation of oxidative DNA damage in myocardial tissue between rats with post-myocardial infarction and sham-operated rats. This up-regulation of activities, initiating the BER pathway, could play an important role during HF by counteracting the effect of genotoxic stress, structural damage of tissue and myocardial remodeling.

Accumulation of mitochondrial DNA damage and bioenergetic dysfunction in CSB defective cells.

Cockayne syndrome (CS) is a complex, progressive disease that involves neurological and developmental impairment and premature aging. The majority of CS patients have mutations in the CSB gene. The CSB protein is involved in multiple DNA repair pathways and CSB mutated cells are sensitive to a broad spectrum of genotoxic agents. We tested the hypothesis that sensitivity to such genotoxins could be mediated by mitochondrial dysfunction as a consequence of the CSB mutation. mtDNA from csb(m/m) mice accumulates oxidative damage including 8-oxoguanine, and cells from this mouse are hypersensitive to the mitochondrial oxidant menadione. Inhibitors of mitochondrial complexes and the glycolysis inhibitor 2-deoxyglucose kill csb(m/m) cells more efficiently than wild-type cells, via a mechanism that does not correlate with mtDNA damage formation. Menadione depletes cellular ATP, and recovery after depletion is slower in csb(m/m) cells. The bioenergetic alteration in csb(m/m) cells parallels the simpler organization of supercomplexes consisting of complexes I, III and IV in addition to partially disassembled complex V in the inner mitochondrial membrane. Exposing wild-type cells to DNA intercalating agents induces complex alterations, suggesting a link between mtDNA integrity, respiratory complexes and mitochondrial function. Thus, mitochondrial dysfunction may play a role in the pathology of CS.