Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

Abstract

DNA is constantly exposed to chemical and environmental mutagens, causing lesions that can stall replication. In order to deal with DNA damage and other stresses, Escherichia coli utilizes the SOS response, which regulates the expression of at least 57 genes, including umuDC. The gene products of umuDC, UmuC and the cleaved form of UmuD, UmuD, form the specialized E. coli Y-family DNA polymerase UmuD 2 C, or polymerase V (Pol V). Y-family DNA polymerases are characterized by their specialized ability to copy damaged DNA in a process known as translesion synthesis (TLS) and by their low fidelity on undamaged DNA templates. Y-family polymerases exhibit various specificities for different types of DNA damage. Pol V carries out TLS to bypass abasic sites and thymine-thymine dimers resulting from UV radiation. Using alanine-scanning mutagenesis, we probed the roles of two active-site loops composed of residues 31 to 38 and 50 to 54 in Pol V activity by assaying the function of single-alanine variants in UV-induced mutagenesis and for their ability to confer resistance to UV radiation. We find that mutations of the N-terminal residues of loop 1, N32, N33, and D34, confer hypersensitivity to UV radiation and to 4-nitroquinoline-N-oxide and significantly reduce Pol V-dependent UV-induced mutagenesis. Furthermore, mutating residues 32, 33, or 34 diminishes Pol V-dependent inhibition of recombination, suggesting that these mutations may disrupt an interaction of UmuC with RecA, which could also contribute to the UV hypersensitivity of cells expressing these variants.Escherichia coli has five DNA polymerases that replicate DNA under different circumstances (22). The replicative polymerase in E. coli is DNA polymerase III (Pol III), a member of the C family. DNA polymerases IV and V are members of the Y family, which specialize in copying damaged DNA in a process known as translesion synthesis (TLS) (22, 47). Y-family DNA polymerases also copy undamaged DNA in an errorprone manner, possibly subjecting DNA to untargeted mutagenesis and potentially leading to antibiotic resistance or cancer (16,17,22,52).E. coli DNA polymerases IV (DinB) and V (UmuDЈ 2 C) are the products of the dinB and umuDC genes, respectively. Due to their potentially mutagenic nature, these proteins are highly regulated in a specific cellular response to DNA damage and other stresses called the SOS response (22,55). The SOS response is initiated when single-stranded DNA (ssDNA) forms downstream from a lesion in DNA due to the inability of the replicative DNA polymerase to copy damaged DNA. RecA then coats the ssDNA to form a RecA-ssDNA nucleoprotein filament, which is the inducing signal for the SOS response. The RecA-ssDNA filament facilitates the autocleavage of LexA, the repressor of the SOS genes, allowing the expression of at least 57 genes (22,66). While many genes are induced in the SOS response, expression of only the umuDC genes is required for SOS mutagenesis (71).The umuDC genes in E. coli encode UmuD, a polymerase manager protein, and UmuC, t...

A Single Residue Unique to DinB-Like Proteins Limits Formation of the Polymerase IV Multiprotein Complex in Escherichia coli

“…It is possible that DinB(C66A) may sequester free UmuD from the cellular pool, which would mimic a deletion of the umuDC operon, previously shown to enhance homologous recombination (15,45,55,56). Thus, if increased MPC formation mediated by DinB(C66A) effectively decreases the concentration of free UmuD, then the increase in homologous recombination is consistent with previous findings (15,45,47). Another possibility is that the increased concentration of DinB MPCs directly enhances recombination.…”

“…We then examined the ability of the dinB(C66A) strain to undergo homologous recombination, which is largely mediated by RecA but is affected by the activities of TLS polymerases (15,45,46). To this end, we employed a P1 transduction assay, which has previously been shown to be effective in quantification of recombination events (47). In this assay, the number of transductants directly correlates with the number of recombination events.…”

Point mutations in Escherichia coli DNA pol V that confer resistance to non-cognate DNA damage also alter protein–protein interactions

“…Cryo-electron microscopy shows that pol V binds to the end as well as within the grooves of the RecA-ssDNA nucleoprotein filament [25]. Our previous work suggested that a possible binding site for RecA on UmuC could be UmuC residues 32 through 34 [26], which was subsequently predicted via molecular modeling [27].…”

“…Strain AB1157 dinB harboring UmuC variants identified in the selection process and re-constructed in fresh plasmids as explained in Section 2.2 (Table 2) was exposed to increasing concentrations of NFZ as described previously [26,49,63]. Serial dilutions (20-fold) of overnight cultures were plated on LB agar plates containing 60 g/mL spectinomycin and the indicated amount of NFZ.…”

“…UV survival and mutagenesis assays were performed as described previously [26,49,63]. Unless otherwise noted, GW8017 harboring plasmids expressing the selected variant were exposed to 25 J/m 2 254 nm UV radiation for mutagenesis assays; results shown are normalized to survival, which varied two-fold or less.…”