PprA Contributes to Deinococcus radiodurans Resistance to Nalidixic Acid, Genome Maintenance after DNA Damage and Interacts with Deinococcal Topoisomerases

PprA is known to contribute to Deinococcus radiodurans' remarkable capacity to survive a variety of genotoxic assaults. The molecular bases for PprA's role(s) in the maintenance of the damaged D. radiodurans genome are incompletely understood, but PprA is thought to promote D. radiodurans's capacity for DSB repair. PprA is found in a multiprotein DNA processing complex along with an ATP type DNA ligase, and the D. radiodurans toposiomerase IB (DraTopoIB) as well as other proteins. Here, we show that PprA is a key contributor to D. radiodurans resistance to nalidixic acid (Nal), an inhibitor of topoisomerase II. Growth of wild type D. radiodurans and a pprA mutant were similar in the absence of exogenous genotoxic insults; however, the pprA mutant exhibited marked growth delay and a higher frequency of anucleate cells following treatment with DNA-damaging agents. We show that PprA interacts with both DraTopoIB and the Gyrase A subunit (DraGyrA) in vivo and that purified PprA enhances DraTopoIB catalysed relaxation of supercoiled DNA. Thus, besides promoting DNA repair, our findings suggest that PprA also contributes to preserving the integrity of the D. radiodurans genome following DNA damage by interacting with DNA topoisomerases and by facilitating the actions of DraTopoIB

PprA promotes <i>D radiodurans</i> resistance to nalidixic acid.

<p>Different dilutions of exponentially growing cells of <i>D. radiodurans</i> (WT), a <i>pprA</i>:<i>cat</i> mutant (Mutant) and the mutant expressing PprA <i>in trans</i> (Mutant +PprA) were spotted on TGY agar plates in absence (Con) and presence of nalidixic acid (Nal) (20 µg/ml) (A). Similarly, these cells were treated with different doses of γ radiation and different dilutions were spotted on TGY agar plate and growth was monitored (B).</p

PprA promotes <i>D. radiodurans</i> growth following DNA damage.

<p><i>D. radiodurans</i> (WT) and a <i>pprA::cat</i> mutant (pprA) were treated with nalidixic acid (20 µg/ml) for 2 h (WTN, pprAN) and γ radiation (6 kGy) (WTG, pprAG). These cells were washed and resuspended in fresh TGY medium, and growth at 30°C was monitored as optical density 600 nm.</p

PprA promotes <i>D. radiodurans</i> genome maintenance following DNA damage.

<p>Both wild type (WT) and <i>pprA</i> mutant (Mutant) <i>D. radiodurans</i> cells were treated with nalidixic acid (Nal) for 2 h and 6 kGy γ radiation (gamma) and stained with DAPI to detect anucleate cells. Representative micrographs are shown in (A), where anucleate cells, lacking DAPI fluorescence, are indicated with arrows. The percent anucleate cells in each condition (∼500 cells/sample) is plotted in (B).</p

Effect of purified PprA on <i>D. radiodurans</i> Type IB topoisomerase activity.

<p>Plasmid DNA having both supercoiled (CC) and relaxed (OC) species was incubated with purified recombinant DraTopoIB (Topo) alone and with increasing molar ratio (1, 2, 3, 4 and 5) of purified PprA. Products were analysed on 1% agarose gels (A). Supercoiled (CC) and relaxed (OC) DNA band intensities were quantified and the ratios of OC to CC were plotted (B). In (C), cell free extracts of <i>E. coli</i> expressing PprA and DraTopoIB from pETpprA and pETtopoIB plasmids respectively were immunoprecipitated with anti PprA antibodies (1). This immunoprecipitate and extracts of cells expressing pETtopoIB (2) and pETpprA (3) independently were immunoblotted with antibodies against (His)6 tag and the sizes of these immunostained bands were compared to the molecular weight marker (M).</p

SMARCAD1 Phosphorylation and Ubiquitination Are Required for Resection during DNA Double-Strand Break Repair

Summary: The chromatin remodeling factor SMARCAD1, an SWI/SNF ATPase family member, has a role in 5′ end resection at DNA double-strand breaks (DSBs) to produce single-strand DNA (ssDNA), a critical step for subsequent checkpoint and repair factor loading to remove DNA damage. However, the mechanistic details of SMARCAD1 coupling to the DNA damage response and repair pathways remains unknown. Here we report that SMARCAD1 is recruited to DNA DSBs through an ATM-dependent process. Depletion of SMARCAD1 reduces ionizing radiation (IR)-induced repairosome foci formation and DSB repair by homologous recombination (HR). IR induces SMARCAD1 phosphorylation at a conserved T906 by ATM kinase, a modification essential for SMARCAD1 recruitment to DSBs. Interestingly, T906 phosphorylation is also important for SMARCAD1 ubiquitination by RING1 at K905. Both these post-translational modifications are critical for regulating the role of SMARCAD1 in DNA end resection, HR-mediated repair, and cell survival after DNA damage. : In Vitro Toxicology Including 3D Culture; Bioengineering; Tissue Engineering Subject Areas: In Vitro Toxicology Including 3D Culture, Bioengineering, Tissue Engineerin

Heat-induced SIRT1-mediated H4K16ac deacetylation impairs resection and SMARCAD1 recruitment to double strand breaks

Hyperthermia inhibits DNA double-strand break (DSB) repair that utilizes homologous recombination (HR) pathway by a poorly defined mechanism(s); however, the mechanisms for this inhibition remain unclear. Here we report that hyperthermia decreases H4K16 acetylation (H4K16ac), an epigenetic modification essential for genome stability and transcription. Heat-induced reduction in H4K16ac was detected in humans, Drosophila, and yeast, indicating that this is a highly conserved response. The examination of histone deacetylase recruitment to chromatin after heat-shock identified SIRT1 as the major deacetylase subsequently enriched at gene-rich regions. Heat-induced SIRT1 recruitment was antagonized by chromatin remodeler SMARCAD1 depletion and, like hyperthermia, the depletion of the SMARCAD1 or combination of the two impaired DNA end resection and increased replication stress. Altered repair protein recruitment was associated with heat-shock-induced γ-H2AX chromatin changes and DSB repair processing. These results support a novel mechanism whereby hyperthermia impacts chromatin organization owing to H4K16ac deacetylation, negatively affecting the HR-dependent DSB repair