Analyzing the Handoff of DNA from UvrA to UvrB Utilizing DNA-Protein Photoaffinity Labeling

DellaVecchia, Matthew J.; Croteau, Deborah L.; Skorvaga, Milan; Dezhurov, Sergey V.; Lavrik, Olga I.; Houten, Bennett Van

doi:10.1074/jbc.m408659200

Cited by 67 publications

(76 citation statements)

References 42 publications

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“…Results of prior studies showed that the UvrABC system can carry out incision of a nicked strand, suggesting that UvrAB recognizes a nick as DNA damage. 20,30 Interestingly, we did not observe loading of UvrB onto nicked DNA using EMSA (data not shown). If UvrB dissociates from a nick during electrophoresis, we would not observe binding by UvrB to nicked DNA using EMSA.…”

Section: Evaluation Of Quantum Dot As a Molecular Pointer For Afm Imamentioning

confidence: 77%

“…Prior studies have shown that this fluorescein adduct can be recognized as a DNA lesion by the NER system. 20,29 A representative agarose gel and quantification of gels from three independent experiments are shown in parts B and C of Figure 3, respectively. In the agarose-EMSA assay, UvrB-DNA complexes ( Figure 3B, lane 3) were clearly resolved, indicating that UvrB was loaded onto damaged DNA by UvrA.…”

Section: Evaluation Of the Dna Binding Function Of Uvrb-qd Using Emsamentioning

confidence: 99%

“…18,19 Once a DNA lesion is encountered, UvrA hands off DNA to UvrB. 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.…”

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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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Section: Evaluation Of Quantum Dot As a Molecular Pointer For Afm Imamentioning

confidence: 77%

Section: Evaluation Of the Dna Binding Function Of Uvrb-qd Using Emsamentioning

confidence: 99%

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

et al. 2008

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“…These mutated amino acid residues, R367A, R289A/R367A, and E307A, are all located on domain 1b oppo- The black bar across the DNA strand indicates that the point of interaction between Phe 527 (F527), and the DNA is several bases downstream (more 3Ј) of the incision site. We have depicted, in various colors, the UvrB amino acid side chains that are thought to be important for DNA damage recognition and processing (5,6,14). A, UvrB has engaged the DNA adduct (red) using Tyr 95 and Tyr 96 (Y95/96).…”

Section: Discussionmentioning

confidence: 99%

“…7. UvrB is believed to first lock the non-damaged strand (14) into place by the interactions between the ␤-hairpin and domain 1b (see Fig. 1).…”

Section: Discussionmentioning

confidence: 99%

Identification of Residues within UvrB That Are Important for Efficient DNA Binding and Damage Processing

Skorvaga

DellaVecchia

Croteau

et al. 2004

Journal of Biological Chemistry

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The UvrB protein is the central recognition protein in bacterial nucleotide excision repair. We have shown previously that the highly conserved ␤-hairpin motif in Bacillus caldotenax UvrB is essential for DNA binding, damage recognition, and UvrC-mediated incision, as deletion of the upper part of the ␤-hairpin (residues 97-112) results in the inability of UvrB to be loaded onto damaged DNA, defective incision, and the lack of stranddestabilizing activity. In this work, we have further examined the role of the ␤-hairpin motif of UvrB by a mutational analysis of 13 amino acids within or in the vicinity of the ␤-hairpin. These amino acids are predicted to be important for the interaction of UvrB with both damaged and non-damaged DNA strands as well as the formation of salt bridges between the ␤-hairpin and domain 1b of UvrB. Nucleotide excision repair (NER)1 is a universal and highly conserved DNA repair pathway found in bacteria and eukaryotic cells. NER is unique because of its broad substrate specificity indicating that the chemical nature of the damage is not solely responsible for damage recognition. Rather a local change in the conformation of the DNA itself resulting from the damage may be required. Damage recognition during NER in bacteria is a multistep process accomplished by the UvrA and UvrB proteins. Recently solved crystal structures of the thermophilic UvrB proteins (1-3) contributed to our understanding of how UvrB may recognize and process DNA lesions.The ␤-hairpin motif of UvrB, which extrudes from domain 1a and contacts domain 1b, is highly conserved in all bacterial species and absolutely required in the process of damage recognition during NER (4). In our padlock binding model for the UvrB⅐DNA preincision complex, we have proposed that the ␤-hairpin is inserted between the two DNA strands, and the non-damaged strand is locked between the ␤-hairpin and domain 1b (3). It is believed that the ␤-hairpin is held in place with respect to domain 1b by two salt bridges and hydrophobic interactions at both the base and the tip of the hairpin (4, 5).The role of the ␤-hairpin in the damage recognition process has been studied by generating the ␤-hairpin deletion and several point mutants and analyzing them using functional assays. We have shown that the ␤-hairpin deletion mutant (⌬␤h) of UvrB still binds to UvrA, forms the UvrAB⅐DNA complex, and has significantly higher ATPase activity compared with WT UvrB. However, the ⌬␤h mutant does not have any strand-separating activity, is not loaded onto the site of the damage, and consequently, does not support incision (4).Goosen and colleagues (5) have probed the role of hydrophobic residues in the ␤-hairpin of E. coli UvrB through a series of double mutants to show that (a) Tyr 92 /Tyr 93 function in damage recognition by preventing UvrB binding to non-damaged sites, (b) Tyr 95 /Tyr 96 at the base of ␤-hairpin have a direct role in damage recognition and are positioned in the vicinity of the lesion in the UvrB⅐DNA complex, and (c) Tyr 101 and Phe 108 in the ...

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UvrA and UvrC subunits of the Mycobacterium tuberculosis UvrABC excinuclease interact independently of UvrB and DNA

2019

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The UvrABC excinuclease plays a vital role in bacterial nucleotide excision repair. While UvrA and UvrB subunits associate to form a UvrA2B2 complex, interaction between UvrA and UvrC has not been demonstrated or quantified in any bacterial species. Here, using Mycobacterium tuberculosis UvrA (MtUvrA), UvrB (MtUvrB) and UvrC (MtUvrC) subunits, we show that MtUvrA binds to MtUvrB and equally well to MtUvrC with submicromolar affinity. Furthermore, MtUvrA forms a complex with MtUvrC both in vivo and in vitro, independently of DNA and UvrB. Collectively, these findings reveal new insights into the pairwise relationships between the subunits of the UvrABC incision complex.

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Analyzing the Handoff of DNA from UvrA to UvrB Utilizing DNA-Protein Photoaffinity Labeling

Cited by 67 publications

References 42 publications

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

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

Identification of Residues within UvrB That Are Important for Efficient DNA Binding and Damage Processing

UvrA and UvrC subunits of the Mycobacterium tuberculosis UvrABC excinuclease interact independently of UvrB and DNA

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