Study protein-protein interaction in Methyl-directed DNA mismatch repair in E. coli: Exonuclease I (Exo I) and DNA helicas II (UvrD) & A Minimal Exonuclease Domain of WRN Forms a Hexamer on DNA and Possesses Both 3’-5’ Exonuclease and 5’-Protruding Strand Endonuclease Activities & Solving the Structure of the Ligand-Binding Domain of the Pregnane-Xenobiotic-Receptor with 17β Estradiol and T1317
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- Study protein-protein interaction in methyl-directed DNA mismatch repair in E. coli: exonuclease I (Exo I) and DNA helicas II (UvrD); A minimal exonuclease domain of WRN forms a hexamer on DNA and possesses both 3’-5’ exonuclease and 5’-protruding strand endonuclease activities; Solving the structure of the ligand-binding domain of the pregnane-xenobiotic-receptor with 17[beta] estradiol and T1317
- Last Modified
- March 21, 2019
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Xue, Yu
- Affiliation: College of Arts and Sciences, Department of Chemistry
- Abstract
- Exonuclease I (ExoI) from Escherichia coli is a monomeric enzyme that processively degrades single stranded DNA in the 3′ to 5′ direction and has been implicated in DNA recombination and repair. It functions in numerous genome maintenance pathways, with particularly well defined roles in methyl-directed mismatch repair (MMR). The Escherichia coli MMR pathway can be reconstituted in vitro with the activities of eight proteins (8). MutS, MutL and MutH are involved in initiation of repair including mismatch recognition and generation of a nick at a nearby GATC sequence (53, 54, 55, 56). The hemimethylated state of GATC sequences immediately following replication serves as a signal to direct repair to the nascent strand of the DNA duplex (57, 58). DNA helicase II and one of several exonucleases (Exonucleas I, Exonuclease VII and RecJ) are required to excise the error-containing DNA strand beginning at the nicked GATC site (34, 35). Restoration of the correct DNA sequence by repair synthesis involves DNA polymerase III holoenzyme and SSB, and the final nick is sealed by DNA ligase (34). To identify interactions with ExoI involved in MMR repair system, we used the yeast two-hybrid system with ExoI as bait. By screening an E.coli genomic library, E. coli DNA helicase II (UvrD) was identified as a potential interacting protein. UvrD has been shown to be required for DNA excision repair, methyl-directed mismatch repair and has some undefined, role in DNA replication and recombination. In this report, in vitro experiments confirm that UvrD and ExoI make a direct physical interaction that may be required for function of the methyl-directed mismatch repair. Werner Syndrome is a rare autosomal recessive disease characterized by a premature aging phenotype, genomic instability and a dramatically increased incidence of cancer and heart disease. Mutations in a single gene encoding a 1,432 amino-acid helicase/exonuclease (hWRN) have been shown to be responsible for the development of this disease. We have cloned, over-expressed and purified a minimal, 171-amino acid fragment of hWRN that functions as an exonuclease. This fragment, encompassing residues 70-240 of hWRN (hWRN-N70-240), exhibits the same level of 3’-5’ exonuclease activity as the previously described exonuclease fragment encompassing residues 1-333 of the full-length protein. The fragment also contains a 5’-protruding DNA strand endonuclease activity at a single-strand/double-strand DNA junction and within singlestranded DNA, as well as a 3’-5’ exonuclease activity on single-stranded DNA. We find hWRN-N70-240 is in a trimer-hexamer equilibrium in the absence of DNA when examined by gel filtration chromatography and atomic force microscopy (AFM). Upon the addition of DNA substrate, hWRN-N70-240 forms a hexamer and interacts with the recessed 3’-end of the DNA. Moreover, we find that the interaction of hWRN-N70-240 with the replication protein PCNA also causes this minimal, 171-amino acid exonuclease region to form a hexamer. Thus, the active form of this minimal exonuclease fragment of human WRN appears to be a hexamer. The implications the results presented here have on our understanding of hWRN’s roles in DNA replication and repair are discussed. The pregnane X receptor (PXR) is a nuclear xenobiotic receptor which acts as a molecular sentry that detects potentially toxic foreign chemicals and activates genes to initiate their breakdown and removal. PXR fills this role by its ability to promiscuously bind to a diverse array of structurally distinct ligands which in turn enables it to activate a wide array of genes such as CYP3A, a monooxygenase involved in breaking down greater than 50 percent of all drugs and MDR1, a drug and xenobiotic efflux pump. Activation of PXR has the potentially deadly side effect of causing drug-drug interactions. Crystal structures of the human PXR ligand binding domain (LBD) have revealed a number of unique features which could facilitate PXR’s promiscuous binding activity. Chief among these is a very large and highly conformable hydrophobic ligand binding cavity. The overall shapes of the ligand binding cavities of hPXR-LBD without ligand and bound to endogenous compound 17β estradiol and the LXR ligand T1317 are distinct. Several structural features of PXR contribute to the plasticity of its binding cavity including an extended beta-sheet region and two novel helices. One of the novel helices and the extended beta-sheet frames the critical second unique helix. This highly flexible helix, called the pseudo-helix due to its variance from the canonical alpha-helical conformation, adopts distinct orientations in every structure solved and plays the single most important role in adapting the shape of the binding cavity to fit different ligand orientations. The accumulating structural data provides important insights into how PXR detects xenobiotics and endobiotics and may prove useful in structure based drug design.
- Date of publication
- December 2008
- DOI
- Resource type
- Rights statement
- In Copyright
- Advisor
- Lord, Susan T.
- Degree granting institution
- University of North Carolina at Chapel Hill
- Language
- Access right
- Open access
- Date uploaded
- October 11, 2010
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