Analysis of the Stimulation of DNA Polymerase V of <i>Escherichia coli</i> by Processivity Proteins

Maor-Shoshani, Ayelet; Livneh, Zvi

doi:10.1021/bi0262909

Cited by 40 publications

(21 citation statements)

References 26 publications

(66 reference statements)

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“…Other protein interactions have also been found to be important for Pol V activity. The ␤ clamp substantially increases processivity in both Pol III and Pol IV in E. coli but increases the processivity of Pol V only 3-to 5-fold (25,35,42,80). The ␤ clamp interacts directly with UmuC utilizing a canonical (4,6,20,41,73) ␤-binding motif, an interaction that is critical for UmuC to participate in TLS (4,6,73).…”

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

2011

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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...

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

2011

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“…Interactions with the ␤ clamp confer high processivity and efficiency on replicative DNA polymerases (22) and increase the processivity of Y family polymerases to various extents (16,25,53). The presence of the ␤ clamp is also thought to play a key role in managing polymerases at the replication fork (15,17).…”

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Characterization of Novel Alleles of the Escherichia coli umuDC Genes Identifies Additional Interaction Sites of UmuC with the Beta Clamp

et al. 2009

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Translesion synthesis is a DNA damage tolerance mechanism by which damaged DNA in a cell can be replicated by specialized DNA polymerases without being repaired. The Escherichia coli umuDC gene products, UmuC and the cleaved form of UmuD, UmuD, comprise a specialized, potentially mutagenic translesion DNA polymerase, polymerase V (UmuD 2 C). The full-length UmuD protein, together with UmuC, plays a role in a primitive DNA damage checkpoint by decreasing the rate of DNA synthesis. It has been proposed that the checkpoint is manifested as a cold-sensitive phenotype that is observed when the umuDC gene products are overexpressed. Elevated levels of the beta processivity clamp along with elevated levels of the umuDC gene products, UmuDC, exacerbate the cold-sensitive phenotype. We used this observation as the basis for genetic selection to identify two alleles of umuD and seven alleles of umuC that do not exacerbate the cold-sensitive phenotype when they are present in cells with elevated levels of the beta clamp. The variants were characterized to determine their abilities to confer the umuDC-specific phenotype UV-induced mutagenesis. The umuD variants were assayed to determine their proficiencies in UmuD cleavage, and one variant (G129S) rendered UmuD noncleaveable. We found at least two UmuC residues, T243 and L389, that may further define the beta binding region on UmuC. We also identified UmuC S31, which is predicted to bind to the template nucleotide, as a residue that is important for UV-induced mutagenesis.

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“…Once loaded, the ␤ clamp slides freely along duplex DNA and functions to tether a variety of different proteins involved in replication and repair to the DNA (4,6,7,9,19,23,24,26,28,(41)(42)(43). The dnaN159 allele encodes a mutant form of the ␤-sliding clamp (␤159) that bears two amino acid substitutions: G66E (glycine at position 66 replaced with glutamic acid) and G174A (14,35,38).…”

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Role of Escherichia coli DNA Polymerase I in Conferring Viability upon the dnaN159 Mutant Strain

et al. 2007

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The Escherichia coli dnaN159 allele encodes a mutant form of the ␤-sliding clamp (␤159) that is impaired for interaction with the replicative DNA polymerase (Pol), Pol III. In addition, strains bearing the dnaN159 allele require functional Pol I for viability. We have utilized a combination of genetic and biochemical approaches to characterize the role(s) played by Pol I in the dnaN159 strain. Our findings indicate that elevated levels of Pol I partially suppress the temperature-sensitive growth phenotype of the dnaN159 strain.In addition, we demonstrate that the ␤ clamp stimulates the processivity of Pol I in vitro and that ␤159 is impaired for this activity. The reduced ability of ␤159 to stimulate Pol I in vitro correlates with our finding that single-stranded DNA (ssDNA) gap repair is impaired in the dnaN159 strain. Taken together, these results suggest that (i) the ␤ clamp-Pol I interaction may be important for proper Pol I function in vivo and (ii) in the absence of Pol I, ssDNA gaps may persist in the dnaN159 strain, leading to lethality of the dnaN159 ⌬polA strain.The Escherichia coli dnaN-encoded ␤-sliding clamp functions as a homodimer and is "loaded" onto primed DNA by the multisubunit DnaX clamp loader complex (5, 21). Once loaded, the ␤ clamp slides freely along duplex DNA and functions to tether a variety of different proteins involved in replication and repair to the DNA (4,6,7,9,19,23,24,26,28,(41)(42)(43). The dnaN159 allele encodes a mutant form of the ␤-sliding clamp (␤159) that bears two amino acid substitutions: G66E (glycine at position 66 replaced with glutamic acid) and G174A (14,35,38). The G174A substitution appears to impair the ability of a hydrophobic cleft in ␤ to interact with the eubacterial clamp-binding motif (QL[S/D]LF) that is conserved in most partner proteins reported to interact with the ␤ clamp (8, 38). E. coli strains bearing the dnaN159 allele display temperature-sensitive growth (14,35,38) and altered DNA polymerase (Pol) usage (29,(38)(39)(40). These phenotypes appear to result, at least in part, from impaired interactions of the mutant ␤159 clamp protein with the replicative DNA polymerase, Pol III (38). As a result of the impaired ␤159-Pol III interaction, E. coli strains bearing the dnaN159 allele display increased utilization of the three SOS-regulated DNA polymerases, Pol II (polB), Pol IV (dinB), and Pol V (umuDC) (29,(38)(39)(40). In contrast to these three Pols, which under certain conditions impede growth of the dnaN159 strain (29, 39, 40), presumably by impairing DNA replication, the catalytic DNA polymerase activity of Pol I (polA) is essential for viability of the dnaN159 strain (38). Importantly, Pol I function is not required by the isogenic dnaN ϩ strain (38), indicating that Pol I plays one or more essential roles in the dnaN159 strain. Since Pol I is a multifunctional protein that participates in DNA replication, as well as numerous DNA repair pathways, several possibilities exist, including Okazaki fragment maturation (21, 31) and single-stranded DNA ...

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Analysis of the Stimulation of DNA Polymerase V of Escherichia coli by Processivity Proteins

Cited by 40 publications

References 26 publications

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

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

Characterization of Novel Alleles of the Escherichia coli umuDC Genes Identifies Additional Interaction Sites of UmuC with the Beta Clamp

Role of Escherichia coli DNA Polymerase I in Conferring Viability upon the dnaN159 Mutant Strain

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