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    Home > Biochemistry News > Biotechnology News > Shanghai Jiaotong University Wu Geng's team collaborated to clarify the structure and function of DndE, an important protein in the archaeal DNA phosphorylation modification pathway

    Shanghai Jiaotong University Wu Geng's team collaborated to clarify the structure and function of DndE, an important protein in the archaeal DNA phosphorylation modification pathway

    • Last Update: 2022-05-18
    • Source: Internet
    • Author: User
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    Recently, Professor Wu Geng's team from the School of Life Science and Technology of Shanghai Jiao Tong University and the State Key Laboratory of Microbial Metabolism collaborated with the team of Professor Wang Lianrong of Wuhan University to elucidate the structure and function of DndE, an important protein in the phosphorylation pathway of archaeal DNA
    .
    The research results were published in the journal mBio under the title "Structural and functional analysis of DndE involved in DNA phosphorothioation in the haloalkaliphilic archaea Natronorubrum bangense JCM10635"

    .
    Prof.
    Wu Geng from the School of Life Science and Technology and Prof.
    Wang Lianrong from Wuhan University are the corresponding authors

    .
    This study for the first time elucidated the structure and function of an archaeal-derived protein DndE that plays an important role in the DNA phosphorylation modification pathway, and deepened people's knowledge and understanding of DNA phosphorylation modification

    .

    In prokaryotes such as bacteria and archaea, a non-bridging oxygen atom on the backbone of DNA is replaced by a sulfur atom, a special epigenetic modification called DNA phosphorylation
    .
    Two types of enzyme systems have been discovered for phosphorothioating modification of DNA: type I DndA-DndB-DndC-DndD-DndE, which performs sulfur modification on double-stranded DNA; and type II SspA-SspB-SspC-SspD , sulfur modification of single-stranded DNA

    .
    DndE belongs to the type I DNA phosphorylation modification system

    .

    In this study, we first analyzed the crystal structure of the DndE protein of the halophilic archaea Natronorubrum bangense JCM10635 strain, and found that, unlike the previously published DndE of E.
    coli B7A strain, which formed a tetramer, the DndE protein of the archaea formed tetramers in crystals and in solution.
    All exist in a single form

    .
    Next, in this study, the purified DndE protein was used to detect DNA nicking (nicking) in vitro, and it was found that, similar to the SspB protein in the type II DNA sulfide modification system SspABCD, DndE has nicking activity on DNA, and covalently closed the ring ( covalently closed circular) One of the single strands of double-stranded DNA is cut, that is, the DNA is nicked, resulting in an open circular DNA, and with the passage of time, a linear DNA is generated

    .
    Moreover, this study also revealed that this DNA nicking activity of DndE is dependent on divalent metal cations, especially magnesium ions, and is independent of the presence or absence of phosphorothioate modifications on DNA

    .
    Run-off sequencing results showed that the nicking activity of DndE on DNA was not DNA sequence specific

    .
    Then, the fluorescence polarization detection experiment using fluorescently labeled DNA and purified DndE protein in vitro shows that DndE protein can directly bind to DNA in vitro, and DndE has the strongest affinity with nicked DNA, and the affinity with double-stranded DNA.
    Second, the affinity to single-stranded DNA is the weakest

    .
    Finally, this study found that mutating the positively charged arginine 19, lysine K23 and arginine R34 on the surface of DndE protein to electrically neutral alanine reduced the affinity between DndE and nicked DNA; The affinity between DndE and nicked DNA is enhanced if glycine 26 of the DndE protein is mutated to a positively charged lysine

    .
    This indicates that DndE protein binds to nicked DNA through its surface positively charged amino acid residues such as arginine 19, lysine K23 and arginine R34

    .

    This article is the structure mechanism of bacteria using SBD domain to recognize sulfur modification on DNA published by Wu Geng's team in Nature Communications in 2018, and the SspB and SspE crystal structures of type II DNA sulfur modification system published in Nature Microbiology in 2020, 2020 Continuation and extension of the crystal structure of the cysteine ​​desulfurase SspA of the type II DNA sulfur modification system published in mBio
    .

    Paper link: https://pubmed.
    ncbi.
    nlm.
    nih.
    gov/35420474/





    School of Life Science and Technology




    School of Life Science and Technology



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