Biology subjects

Sass, T. H.

Publications and source records attributed to Sass, T. H..

2 recordsLinked to original sources

bioRxiv2021

DnaA and SspA Regulation of the iraD gene of E. coli: an alternative DNA damage response independent of LexA/RecA

The transcription factor RpoS ({sigma}S) of Escherichia coli controls a large number of genes important for tolerance of a variety of stress conditions. IraD promotes the post-translation stability of RpoS by inhibition of RssB, an adaptor protein for ClpXP degradation. We have previously documented DNA damage induction of iraD expression, independent of the SOS response. Both iraD and rpoS are required for tolerance to DNA damaging treatments such as H2O2 and the replication inhibitor azidothymidine in the log phase of growth. Using luciferase gene fusions to the 672 bp iraD upstream region, we show here that both promoters of iraD are induced by AZT. Genetic analysis suggests that both promoters are repressed by DnaA-ATP, partially dependent on a putative DnaA box at -81 bp, and regulated by RIDA (regulatory inactivation of DnaA), dependent on the DnaN processivity clamp. By electrophoretic mobility shift assays we show that purified DnaA protein binds to the iraD upstream region, so DnaA regulation of IraD is likely to be direct. DNA damage induction of iraD during log phase growth is abolished in the dnaA-T174P mutant, suggesting that DNA damage, in some way, relieves DnaA repression, possibly through the accumulation of replication clamps and enhanced RIDA. We demonstrate that the RNA-polymerase associated factor, SspA (stringent starvation protein A), induced by the accumulation of ppGpp, also affects IraD expression, with a positive effect on constitutive expression and a negative effect on AZT-induced expression, in a fashion independent of DnaA. SIGNIFICANCEDNA damage can lead to cell death or genomic instability. Cells have evolved transcriptional responses that sense DNA damage and up-regulate tolerance and repair factors; the LexA/RecA-regulated SOS response in E. coli was the first example of such a system. This work describes an alternative DNA damage response, controlled by DnaA and the IraD post-translational regulator of RpoS. The cellular signals for this response, we propose, are empty replication processivity ({beta}) clamps that accumulate at replication blocks. IraD expression is also regulated by stringent starvation protein, SspA, induced by nutrient deprivation. This SOS-independent DNA damage response integrates a signal of incomplete replication with starvation to modulate expression of genes that promote the completion of replication.

genetics↗

bioRxiv2021

Mutational analysis of DinG-family helicase YoaA: its expression and interaction with replication clamp-loader protein HolC in E. coli

The XP-D/DinG family of DNA helicases contribute to genomic stability in all three domains of life. We investigate here the role of one of these proteins,YoaA, of Escherichia coli. In E. coli,YoaA aids tolerance to the nucleoside azidothymidine (AZT), a DNA replication inhibitor and physically interacts with a subunit of the DNA polymerase III holoenzyme, HolC. We map the residues of YoaA required for HolC interaction to its C-terminus by yeast two-hybrid analysis. We propose that this interaction competes with HolCs interaction with HolD and the rest of the replisome;YoaA indeed inhibits growth when overexpressed, dependent on this interaction region. By gene fusions we show YoaA is repressed by LexA and induced in response to DNA damage as part of the SOS response. Induction of YoaA by AZT is biphasic with an immediate response after treatment and a slower response that peaks in the late log phase of growth. This growth-phase dependent induction by AZT is not blocked by lexA3 (Ind-), which normally negates its self-cleavage, implying another means to induce the DNA damage response that responds to the nutritional state of the cell. We propose that YoaA helicase activity increases access to the 3 nascent strand during replication; consistent with this,YoaA appears to aid removal of potential A-to-T transversion mutations in ndk mutants, which are prone to nucleotide misincorporation. YoaA and its paralog DinG also may initiate template-switching that leads to deletions between tandem repeats in DNA. IMPORTANCEMaintaining genomic stability is crucial for all living organisms. Replication of DNA frequently encounters barriers that must be removed to complete genome duplication. Balancing DNA synthesis with its repair is critical and not entirely understood at a mechanistic level.The YoaA protein, studied here, is required for certain types of DNA repair and interacts in an alternative manner with proteins that catalyze DNA replication. YoaA is part of the well-studied LexA-regulated response to DNA damage, the SOS response. We describe an unusual feature of its regulation that promotes induction after DNA damage as the culture begins to experience starvation. Replication fork repair integrates both DNA damage and nutritional signals. We also show that YoaA affects genomic stability.

genetics↗