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

Abstract

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

UmuD2 Inhibits a Non-covalent Step during DinB-mediated Template Slippage on Homopolymeric Nucleotide Runs

“…Moreover, the ϳ0.1 mM UmuD 2 concentration used in our assays may alter its protein folding. UmuD 2 is an intrinsically disordered protein displaying a CD spectrum characteristic of a random coil at low concentrations (ϳ5 M) and a spectrum indicating the presence of a ␤-sheet at high concentrations (ϳ1 mM) (31). This suggests multiple protein concentration-dependent conformations, only a subset of which may be competent to bind and inhibit DinB catalysis.…”

“…Native UmuD was purified as described previously (31). RecA protein and T4-polynucleotide kinase were purchased from New England Biolabs.…”

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

“…In both UmuD and UmuDЈ, G129 appears to be mostly buried and D84 appears to be partially buried and involved in main chain and side chain hydrogen bonding interactions with S81. Intriguingly, G129 does not appear to be near the active site, involved in interactions with the N-terminal arms, or near the dimer interface, although mutations in this residue might decrease dimer formation (see Discussion) (28,42).…”

“…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 .…”

“…The presence of the ␤ clamp is also thought to play a key role in managing polymerases at the replication fork (15,17). UmuD and UmuDЈ interact with the ␤ clamp physically, as well as genetically (5,42,46,48,49). Deletion of the N-terminal 9 residues of UmuD has little effect on its ability to crosslink to the ␤ clamp, while deletion of the N-terminal 19 residues results in a dramatic decrease in, but not a complete loss of, cross-linking efficiency (48).…”