An Active Site Aromatic Triad in Escherichia coli DNA Pol IV Coordinates Cell Survival and Mutagenesis in Different DNA Damaging Agents

Godoy VG (2011) An Active Site Aromatic Triad in Escherichia coli DNA Pol IV Coordinates Cell Survival and Mutagenesis in Different DNA Damaging Agents. PLoS ONE 6(5): e19944. doi:10.1371/journal.pone.0019944 An Active Site Aromatic Triad in Escherichia coli DNA Pol IV Coordinates Cell Survival and Mutagenesis in Different DNA Damaging Agents Ryan W. Benson 0 Matthew D. Norton 0 Ida Lin 0 William S. Du Comb 0 Veronica G. Godoy 0 Martin G. Marinus, University of Massachusetts Medical School, United States of America 0 Department of Biology, Northeastern University , Boston, Massachusetts , United States of America DinB (DNA Pol IV) is a translesion (TLS) DNA polymerase, which inserts a nucleotide opposite an otherwise replicationstalling N2-dG lesion in vitro, and confers resistance to nitrofurazone (NFZ), a compound that forms these lesions in vivo. DinB is also known to be part of the cellular response to alkylation DNA damage. Yet it is not known if DinB active site residues, in addition to aminoacids involved in DNA synthesis, are critical in alkylation lesion bypass. It is also unclear which active site aminoacids, if any, might modulate DinB's bypass fidelity of distinct lesions. Here we report that along with the classical catalytic residues, an active site ''aromatic triad'', namely residues F12, F13, and Y79, is critical for cell survival in the presence of the alkylating agent methyl methanesulfonate (MMS). Strains expressing dinB alleles with single point mutations in the aromatic triad survive poorly in MMS. Remarkably, these strains show fewer MMS- than NFZ-induced mutants, suggesting that the aromatic triad, in addition to its role in TLS, modulates DinB's accuracy in bypassing distinct lesions. The high bypass fidelity of prevalent alkylation lesions is evident even when the DinB active site performs errorprone NFZ-induced lesion bypass. The analyses carried out with the active site aromatic triad suggest that the DinB active site residues are poised to proficiently bypass distinctive DNA lesions, yet they are also malleable so that the accuracy of the bypass is lesion-dependent. - Funding: This work was supported by the 1RO1GM088230-01A1 award from NIGMS to V. G. Godoy and in part by an RSFD award to V. G. Godoy by the office of the Provost of Northeastern University (2008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Replicative DNA polymerases are multi-protein complexes responsible for synthesizing a high fidelity copy of a cells genome. Persistent lesions on the template DNA, which DNA repair pathways have failed to recognize, result in stalling of DNA replication, a potentially lethal event [1]. To avoid lethality, specialized DNA polymerases insert deoxynucleotides (dNTPs) opposite replication-blocking DNA lesions in a process known as translesion synthesis (TLS). This is largely a low fidelity process usually resulting in elevated mutagenesis [1,2]. In Escherichia coli there are three TLS polymerases that are regulated by the SOS gene network, one of the cellular responses to DNA damage and environmental stress [1,3]. The polB gene encodes the B family DNA Pol II, while the dinB gene and the umuDC operon encode the two Y family DNA polymerases, DNA Pol IV and DNA Pol V respectively [1,4,5,6,7]. DinB is of particular interest because of its evolutionary conservation [1,6,8] and its high basal intracellular concentration (,250 nM) [1,9,10]. Indeed, this is approximately 17 fold higher [10] than that of DNA Pol III complex (the replicative DNA polymerase, 15 nM; [9]) and is similar to that of the processivity clamp (b-clamp, 250 nM; [11,12]), an essential replication factor known to both recruit all DNA polymerases to the replication fork and manage their activity in the cell [13,14]. E.coli cells lacking the dinB gene (DdinB) are sensitive to nitrofurazone (NFZ) and 4-nitroquinoline-1-oxide (4-NQO) [15,16], reagents that create persistent DNA lesions on the N2 group of deoxyguanosine (N2-dG) [17,18]. Recent evidence suggests that DinB and its homologues can also perform TLS of lesions that are the product of alkylation of DNA bases [19,20,21]. Alkylating agents are both a byproduct of the cells metabolism and also come from diverse exogenous sources generating DNA damage in prokaryotic and eukaryotic cells [1,22,23,24,25]. In addition, alkylating agents are used as anti-cancer chemotherapeutic agents, [1,26,27], underscoring the significance of understanding the cellular mechanisms of alkylation lesion tolerance. It is known that base excision repair pathways are the primary cellular response to alkylation damage [1,28,29,30,31,32], though Y family DNA polymerases are also part of this response [1,19,20,21]. These polymerases likely bypass 3-methyladenine (3-meA; [1,19,20,21,33,34]), a prevalent alkylation lesion that persists on the DNA and brings about replication fork stalling and cell death [20,21,35,36]. Indeed, E. coli strains lacking the dinB gene (DdinB) are sensitive to several alkylating agents, such as methyl methanesulfonate (MMS; [19]). Similar sensitivity is found in eukaryotic cells deficient in TLS polymerases [20,21]. Thus, the evidence so far indicates that if DNA repair pathways do not effectively recognize 3-meA, Y family DNA polymerases are critical in the cells response to alkylation damage [19,20,21]. Unfortunately, 3-meA has a very short in vitro half-life [21,37], making difficult to directly investigate the bypass mechanisms of this alkylation lesion. Most of our knowledge in regard to the active site of DinB has been acquired through studies with reagents that generate N2-dG lesions [15,16]. However, it is not known which aminoacids in the DinB active site are important for the bypass of alkylation lesions, e.g. most likely 3-meA. It is also unclear whether the same active site residues are involved in the bypass or its fidelity of both alkylation and N2-dG lesions. Structural modeling predicts that Pol k (the mammalian DinB homologue) could accommodate either the N2-dG or 3-meA minor groove adducts in its active site in a conformation that would allow both insertion and extension from either adduct [20]. We studied a triad of aromatic residues (Fig. 1) that is conserved in Y family DNA polymerases including Pol g and Pol k [38,39] and used as a control the strictly catalytic aspartic acid 103 (D103). This is known to be critical for DNA synthesis and thus unable to complement a DdinB strain [16,40,41]. Here, we describe the analysis of the aromatic triad residues of DinB in response to DNA damage generated by treatment with MMS or NFZ, reagents that create respectively alkylation or N2-dG lesions in vivo. This report describes the effect of changing the aromatic triad residues to those of different polarity or size on both survival and DNA damag (...truncated)