A novel role for the transcriptional modulator NusA in DNA repair/damage tolerance pathways in Escherichia coli

Abstract

All organisms must contend with the consequences of DNA damage, induced by a variety of both endogenous and exogenous sources. Mechanisms of DNA repair and DNA damage tolerance are crucial for cellular survival after DNA damage. Translesion DNA synthesis (TLS) is one such mechanism of DNA damage tolerance which utilizes a specialized translesion DNA polymerase capable of catalyzing DNA synthesis on imperfect templates. There are two TLS polymerases present in Escherichia coli encoded by the dinB (Pol IV) and umuDC (Pol V) gene products. While TLS polymerases provide a variety of benefits to the cell, it is important that they are properly regulated as they have reduced fidelity on undamaged DNA compared to replicative DNA polymerases. Here I present evidence that the essential transcriptional modulator NusA associates with TLS polymerases in E. coli both physically, as noted for DinB, and genetically, with DinB and the umuDC gene products. Mutation of nusA renders cells sensitive to DNA damaging agents and produces phenotypes reminiscent of mutants with altered DNA processing. Moreover, I report that the nusAll mutation completely eliminates the formation of adaptive mutants, revealing that nusA+ function is required for cells to adapt and mutate in response to stress. Though the phenomenon of adaptive mutagenesis also requires dinB+, my data suggest that the role for nusA in adaptive mutagenesis extends beyond an interaction with DinB.

(cont.) Furthermore, I report that NusA in addition to having a role in transcription elongation is also important for promoting survival after DNA damage. Phenotypes of nusA mutants are more exaggerated than those of TLS polymerase mutants. Genetic interactions of nusA+ with the nucleotide excision repair pathway suggest that nusA+ may play a role in a new class of NusA-dependent transcription coupled repair. Moreover, I have isolated RNA polymerase mutants with altered ability to survive after DNA damage, and this altered ability is absolutely dependent on nusA+ and uvrA+. The completion of translesion DNA synthesis requires both the insertion of a nucleotide opposite the adducted template base and extension from that position by several subsequent nucleotide additions. We present evidence that DinB is specialized to perform strikingly proficient extension after insertion opposite an N2-dG lesion. Our data indicate that cellular survival is coupled to completion of TLS and regulation of these precise steps in vivo is genetically complex and involves the toxin-antitoxin module MazEF and the iron import protein TonB.