Function of the homologous regions of the Escherichia coli DNA excision repair proteins UvrB and UvrC in stabilization of the UvrBC–DNA complex and in 3′-incision

Moolenaar, Geri F.; Franken, Kees L. M. C.; Putte, Pieter van de; Goosen, Nora

doi:10.1016/s0921-8777(97)00042-6

Cited by 39 publications

(51 citation statements)

References 29 publications

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“…The distribution of the methionine residues in Bca UvrB is illustrated in Figure 1a. The assigned 1 H-13 C HSQC spectrum of the methionine methyl region of the spectrum of UvrB labeled with [methyl- 13 C]methionine is shown in Figure 2a. Assignments of most of the methyl resonances were made based on the construction of site-directed methionine to leucine mutants (Table 1).…”

Section: Nmr Studies Of Site-directed Mutantsmentioning

confidence: 99%

“…Although TEMPOL-induced broadening has previously been used to evaluate the solvent accessibility of methionine residues in [methyl- 13 C]methionine-labeled calmodulin 30 , it was anticipated that the substantial variability of peak intensity in the labeled UvrB ( Figure 2a) would limit the utility of this approach. Unexpectedly, addition of 20 mM TEMPOL to the UvrB M296L mutant (used due to its availability), resulted in both 13 C shift and relaxation perturbations ( Figure 6). In surface accessibility studies, TEMPOL-induced shifts were concluded to be small by Petros et al 28 .…”

Section: Solvent Accessibility -Response To Tempolmentioning

confidence: 99%

“…1 H-13 C HSQC of [methyl- 13 C] UvrB M296L mutant before and after the addition of 20 mM TEMPOL are shown in Figure 6, and several 1D slices derived from this spectrum are included as Supplementary Material-S2. In order to facilitate comparison of the extensively overlapped resonances, the spectrum of the TEMPOL-containing sample has been offset by 0.15 ppm in the 1 H dimension only.…”

Section: Solvent Accessibility -Response To Tempolmentioning

confidence: 99%

“…In order to characterize the solution behavior of the 75 kD UvrB protein, we have introduced [methyl- 13 C]methionine into Bca UvrB, thereby providing NMR probes that are distributed throughout the molecule. In general, introduction of labeled methionine residues provides an ideal NMR labeling strategy for studies of large biological molecules due to the three equivalent methyl protons and the very low order parameters that characterize methionine methyl groups 19-21 , resulting in narrow linewidths even for very large macromolecules.…”

Section: Introductionmentioning

confidence: 99%

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NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB–domain 4 heterodimer formation in solution

DellaVecchia

Merritt

et al. 2007

Journal of Molecular Biology

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SummaryUvrB is a central DNA damage recognition protein involved in bacterial nucleotide excision repair (NER). Structural information has been limited by the apparent disorder of the C-terminal domain 4 in crystal structures of intact UvrB; in solution, the isolated domain 4 is found to form a helix-loophelix dimer. In order to gain insight into the solution behavior of UvrB, we have performed NMR studies on [methyl-13 C]methionine-labeled UvrB from B. caldotenax (MW = 75 kD). The 13 methyl resonances were assigned on the basis of site-directed mutagenesis and domain deletion. Solvent accessibility was assessed based on the relaxation and chemical shift responses of the probe methyl resonances to the stable nitroxide, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPOL). Methionine 632, located at the potential dimer interface of domain 4, provides an ideal probe for UvrB dimerization behavior. The M632 resonance of UvrB is very broad, consistent with some degree of monomer-dimer exchange, and/or conformational instability of the exposed dimer interface. Upon addition of unlabeled domain 4 peptide, the M632 resonance of UvrB sharpens and shifts to a position consistent with a UvrB-domain 4 heterodimer. A K D value of 3.3 μM for the binding constant of UvrB with the domain 4 peptide was derived from surface plasmon resonance studies. Due to the flexibility of the domain 3-4 linker, inferred from limited proteolysis data and from the relaxation behavior of linker residue M607, the position of domain 4 is constrained not by the stiffness of the linking segment, but by direct interactions with domains 1-3 in UvrB. In summary, UvrB homodimerization is disfavored, while domain 4 homodimerization and UvrB-domain 4 heterodimerization are allowed.

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Section: Nmr Studies Of Site-directed Mutantsmentioning

confidence: 99%

Section: Solvent Accessibility -Response To Tempolmentioning

confidence: 99%

Section: Solvent Accessibility -Response To Tempolmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB–domain 4 heterodimer formation in solution

DellaVecchia

Merritt

et al. 2007

Journal of Molecular Biology

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“…20 UvrB then verifies the damage and recruits the endonuclease UvrC, which carries out incisions on the damaged DNA strand. [21][22][23] In this report, we use AFM imaging to demonstrate the successful conjugation of single quantum dots to UvrB. We show that an agarose gel-based electrophoresis mobility shift assay (EMSA) can be used to evaluate the DNA binding function of UvrB-QD conjugates.…”

mentioning

confidence: 99%

Functional Characterization and Atomic Force Microscopy of a DNA Repair Protein Conjugated to a Quantum Dot

et al. 2008

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Quantum dots (QDs) possess highly desirable optical properties that make them ideal fluorescent labels for studying the dynamic behavior of proteins. However, a lack of characterization methods for reliably determining protein-quantum dot conjugate stoichiometry and functionality has impeded their widespread use in single-molecule studies. We used atomic force microscopic (AFM) imaging to demonstrate the 1:1 formation of UvrB-QD conjugates based on an antibodysandwich method. We show that an agarose gel-based electrophoresis mobility shift assay and AFM can be used to evaluate the DNA binding function of UvrB-QD conjugates. Importantly, we demonstrate that quantum dots can serve as a molecular marker to unambiguously identify the presence of a labeled protein in AFM images.In recent years, quantum dot (QD) bioconjugates have become increasingly popular in fluorescence experiments due to their narrow spectral emission width, strong emission intensity, small size, and good photostability. 1,2 Their strong emission intensities allow single quantum dots to be visualized by epifluorescence microscopy, while individual molecules of green fluorescent protein (GFP) and other synthetic fluorophores require the use of total internal reflection fluorescence (TIRF) microscopy to enhance the signal-tonoise ratio. 3 The unique properties of quantum dots also enable long-term tracking and monitoring of fast dynamics in single-molecule fluorescence microcopy studies. However, applications of protein-QD conjugates have so far been limited to antibodies for cell imaging, Western blot analysis, and fluorescence in situ hybridization. [4][5][6][7][8] Enzymatic studies of proteins using quantum dot conjugates have been limited to only a few proteins such as myosin, dynein, actin filaments, Rdh54, and Msh2-Msh6. 9-14 Several barriers are commonly encountered when using quantum dot labeled proteins in single-molecule studies. For example, conjugation of a quantum dot to a protein can potentially interfere with protein-protein and protein-ligand interactions. In addition, it is possible to conjugate more than one protein to a single functionalized quantum dot. Although gel electrophoresis in Supporting Information Available: Additional information includes Materials and Methods as well as figures showing more than one particles attached to a quantum dot at a 1:1 ratio of QD:UvrB-Ab, the aggregation of quantum dots in the presence of biotinylated HA antibody, assays confirming the nicking of the 517 base pair PCR fragment, statistical analysis of position distributions of UvrA and UvrB-QD on non-nicked DNA, and examples of UvrB-QD and UvrA-UvrB-QD conjugates on DNA. Atomic force microscopy (AFM) is a powerful single-molecule technique for studying biomolecular interactions. This technique can produce topographic images at high resolution (typically ≥10 nm). 16,17 However, for more complex, heteromeric assemblies, which are ubiquitous in many biological processes, using AFM imaging, we cannot always distinguish between different ...

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Functional Domains of the E. coli UvrABC Proteins in Nucleotide Excision Repair

et al. 1998

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Function of the homologous regions of the Escherichia coli DNA excision repair proteins UvrB and UvrC in stabilization of the UvrBC–DNA complex and in 3′-incision

Cited by 39 publications

References 29 publications

NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB–domain 4 heterodimer formation in solution

NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB–domain 4 heterodimer formation in solution

Functional Characterization and Atomic Force Microscopy of a DNA Repair Protein Conjugated to a Quantum Dot

Functional Domains of the E. coli UvrABC Proteins in Nucleotide Excision Repair

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