A Non-cleavable UmuD Variant That Acts as a UmuD′ Mimic

Beuning, Penny J.; Simon, Sharotka M.; Zemła, Adam; Barsky, Daniel; Walker, Graham C.

doi:10.1074/jbc.m511101200

Cited by 26 publications

(99 citation statements)

References 49 publications

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“…Although no high-resolution structural data are available for UmuD 2 , we have recently proposed four energy-minimized symmetrical models of UmuD 2 (15). Previous single-cysteine studies of UmuD 2 , which probed the structure of UmuD 2 in solution at physiologically relevant concentrations, have generally been consistent with our structural models.…”

supporting

confidence: 51%

“…Interestingly, some positions that are predicted to be far away from the dimer interface also cross-link (16,18). However, these residues can come together if the N-terminal arms are in an intermediate conformation (15). These results suggest that UmuD 2 may interchange among multiple conformations in solution.…”

mentioning

confidence: 77%

“…Because both the ␤-subunit and DinB have a more typical secondary structure than UmuD 2 (data not shown), it is likely that the increase in secondary structure of the complex is mostly due to an increased ␤-sheet content in UmuD 2 . However, we cannot rule out the possibility that binding of UmuD 2 may cause a conformational change in the interacting proteins as well (7,15). evidence are inconsistent with monomeric umuD gene products at physiological concentrations.…”

Section: Crowding Agents and Specific Protein-protein Interactions Inmentioning

confidence: 99%

“…We have previously shown that UmuD 2 interacts with DinB (K D ϭ 0.64 M) (7) and with the ␤-subunit of DNA Pol III (K D ϭ 5.5 M) (15). To test whether these interactions induce secondary structure in UmuD 2 at M concentrations, we took the CD spectrum of 50 M UmuD 2 in the presence of 50 M interacting protein.…”

Section: Crowding Agents and Specific Protein-protein Interactions Inmentioning

confidence: 99%

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Regulation of Escherichia coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products

Simon

Sousa²,

Mohana‐Borges³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Products of the umuD gene in Escherichia coli play key roles in coordinating the switch from accurate DNA repair to mutagenic translesion DNA synthesis (TLS) during the SOS response to DNA damage. Homodimeric UmuD 2 is up-regulated 10-fold immediately after damage, after which slow autocleavage removes the Nterminal 24 amino acids of each UmuD. The remaining fragment, UmuD 2, is required for mutagenic TLS. The small proteins UmuD2 and UmuD 2 make a large number of specific protein-protein contacts, including three of the five known E. coli DNA polymerases, parts of the replication machinery, and RecA recombinase. We show that, despite forming stable homodimers, UmuD 2 and UmuD 2 have circular dichroism (CD) spectra with almost no ␣-helix or ␤-sheet signal at physiological concentrations in vitro. High protein concentrations, osmolytic crowding agents, and specific interactions with a partner protein can produce CD spectra that resemble the expected ␤-sheet signature. A lack of secondary structure in vitro is characteristic of intrinsically disordered proteins (IDPs), many of which act as regulators. A stable homodimer that lacks significant secondary structure is unusual but not unprecedented. Furthermore, previous single-cysteine cross-linking studies of UmuD 2 and UmuD 2 show that they have a nonrandom structure at physiologically relevant concentrations in vitro. Our results offer insights into structural characteristics of relatively poorly understood IDPs and provide a model for how the umuD gene products can regulate diverse aspects of the bacterial SOS response.natively unfolded ͉ SOS response ͉ unstructured ͉ denatured ͉ DNA repair

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supporting

confidence: 51%

mentioning

confidence: 77%

Section: Crowding Agents and Specific Protein-protein Interactions Inmentioning

confidence: 99%

Section: Crowding Agents and Specific Protein-protein Interactions Inmentioning

confidence: 99%

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Regulation of Escherichia coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products

Simon

Sousa²,

Mohana‐Borges³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…UmuC activity is regulated by the accessory protein UmuD. UmuD is a homodimeric protein composed of a C-terminal globular domain and N-terminal arms (5,12,35,42,47). After SOS induction, UmuD initially persists in the full-length dimeric form, UmuD 2 .…”

mentioning

confidence: 99%

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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Translesion DNA Synthesis

2005

Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine

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All living organisms are continually exposed to agents that damage their DNA, which threatens the integrity of their genome. As a consequence, cells are equipped with a plethora of DNA repair enzymes to remove the damaged DNA. Unfortunately, situations nevertheless arise where lesions persist, and these lesions block the progression of the cell's replicase. Under these situations, cells are forced to choose between recombination-mediated "damage avoidance" pathways, or use a specialized DNA polymerase (pol) to traverse the blocking lesion. The latter process is referred to as Translesion DNA Synthesis (TLS). As inferred by its name, TLS not only results in bases being (mis)incorporated opposite DNA lesions, but also downstream of the replicase-blocking lesion, so as to ensure continued genome duplication and cell survival. Escherichia coli and Salmonella typhimurium possess five DNA polymerases, and while all have been shown to facilitate TLS under certain experimental conditions, it is clear that the LexA-regulated and damage-inducible pols II, IV and V perform the vast majority of TLS under physiological conditions. Pol V can traverse a wide range of DNA lesions and performs the bulk of mutagenic TLS, whereas pol II and pol IV appear to be more specialized TLS polymerases. HISTORICAL OVERVIEWWe now know that TLS is largely facilitated by specialized DNA polymerases that can accommodate bulky adducts in their active sites. Such properties are usually associated with a dramatic decrease in replication fidelity and as a consequence, TLS is often error-prone, leading to an increase in cellular mutagenesis. Significant progress in understanding the

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A Non-cleavable UmuD Variant That Acts as a UmuD′ Mimic

Cited by 26 publications

References 49 publications

Regulation of Escherichia coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products

Regulation of Escherichia coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products

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

Translesion DNA Synthesis

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