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    Home > Biochemistry News > Biotechnology News > Using super-resolution imaging technology to reveal a new mechanism for DNA replication initiation site selection

    Using super-resolution imaging technology to reveal a new mechanism for DNA replication initiation site selection

    • Last Update: 2021-07-31
    • Source: Internet
    • Author: User
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      There are approximately 250,000 sites in the human cell genome that can be used to initiate DNA replication.
    These sites are called DNA replication initiation sites

    .
    Interestingly, a cell uses only about one-tenth of it to complete DNA replication in a cell cycle, and its selection of the replication start site shows a certain regularity, which is reflected in the different replication start sites.
    Relatively conservative replication initiation probability

    .
    Although previous studies have found that the activity of the replication initiation site is related to a variety of genetic and epigenetic factors (Figure 1 above), there has been a lack of consensus on the mechanism of replication initiation site selection

    .

     

      Among them, regarding the relationship between the selection of the DNA replication initiation site and the structure of chromatin, the predecessors in this field inferred a model of DNA replication through some experimental results: it is believed that at the beginning of DNA replication, in the chromatin domain (DNA In the replication domain or topological association domain (TAD), the DNA replication start sites located inside the domain and clustered together have a higher initiation probability (Figure 1 below).
    This clustering initiates DNA replication is conducive to replication.
    Collaborate and improve replication efficiency

    .

      

      

      Figure 1: (top) The activity of the DNA replication start site is related to many factors, Michalis et al.
    NATURE REVIEWS (2015); (bottom) a traditional model of the DNA replication start site selection mechanism

    .
    This model indicates that the replication initiation sites (red) clustered inside the chromatin domain during the G1/S period have a higher probability of being used (indicated by a six-pointed star) to initiate DNA replication

    .

      

      However, this model lacks direct imaging evidence due to technical barriers, including how to accurately label the chromatin domain and its origin of replication based on sequence information at the same time, and compare the domain (radius 200-300 nm) and origin of replication.
    Realize precise imaging

    .
    On July 13, 2021, Peking University Future Technology College and Peking University Biomedical Frontier Innovation Center (BIOPIC) Sun Yujie's research group published a research paper entitled Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency in 
    Genome Biology  online , Through the hybridization and labeling of the DNA probe library, the precise labeling of the chromatin domain (green in Figure 2) and its replication initiation site (purple in Figure 2) was achieved, and super-resolution microscopy (STORM) ) For accurate positioning .
    The results show that at the beginning of DNA replication (G1/S time point), the replication initiation sites (ORI2, ORI3) used with high probability are distributed in the periphery of the chromatin domain, while the replication initiation sites used with low probability are distributed in the periphery of the chromatin domain.
    The dots (ORI4, ORI5) are distributed inside the chromatin domain
    .
    At the same time, the site of the initial replication using metabolic markers is also on the periphery of the replication domain.
    The above results directly prove that the traditional model (Figure 1 bottom) is not accurate
    .



      

      

      

      

      Figure 2: The distribution of replication origin sites (purple) in the TAD (green) chromatin topological association structure domain at different periods
    .
    The
    overlap of green and purple is shown in white .
    The scale bar is 200 nm

    .

      

      At the same time, the researchers found that the spatial distribution of the replication start site in the domain is relatively random when the DNA in the pre-middle stage of G1 has not started to replicate
    .
    Interestingly, some replication initiation sites have undergone a spatial shift from G1 to entering S phase, and this spatial shift makes them more likely to be used to initiate replication than other replication initiation sites

    .
    In order to study the changes in the spatial structure of chromatin and the mechanism that affects DNA replication, researchers first explored the reasons for the changes in the spatial structure of chromatin

    .
    They found that reducing the expression of CTCF or cohesin, or inhibiting transcription, will cause the spatial distribution of replication start sites in the G1/S phase to become relatively random

    .
    This means that it is very likely that the transcription process affects the chromatin structure and thus changes the spatial distribution of the replication start site in the chromatin domain

    .
    Finally, the researchers found that unlike MCM, CTCF and other proteins can enter the domain, and the DNA replication complex protein PCNA will form clusters and distribute on the periphery of the domain

    .
    Therefore, the replication initiation sites distributed on the periphery of the domain have more chances to be used to initiate DNA replication

    .

      

      Combining the above results, the researchers proposed the "Chromatin Re-organization Induced Selective Initiation" (CRISI) model (Figure 3)
    .
    In the early and mid-G1 phases, the origin of replication is relatively randomly distributed in the domain

    .
    The progress of the G1 transcription process changes the local chromatin structure, resulting in a change in the distribution of replication initiation sites.
    The replication initiation sites exposed on the periphery of the structure domain preferentially bind to the peripheral DNA replication complex and initiate replication

    .
    Importantly, this discovery not only reveals the importance of the three-dimensional structure of chromatin in the regulation of DNA replication, but also reconciles the various genetic and epigenetic factors discovered by multiple research groups (Figure 1 above).
    The link between transcription, chromatin local structure and DNA replication site selection

    .

      

      

      Figure 3: The "Chromatin Re-organization Induced Selective Initiation" (CRISI) model establishes the relationship between the selection of the DNA replication initiation site and the changes in the chromatin structure of transcriptional regulation
    .

      

      Researcher Sun Yujie, School of Future Technology, Peking University and Peking University Biomedical Frontier Innovation Center (BIOPIC), is the corresponding author of the research paper
    .
    The
    first author of this article is Li Yongzheng and Xue Boxin graduated from the Biomedical Frontier Innovation Center of Peking University, and third-year doctoral student Zhang Mengling
    .
    This research was supported and helped by the team of researcher Li Guohong from the Institute of Biophysics

    .

      

      Paper link:

      https://genomebiology.
    biomedcentral.
    com/articles/10.
    1186/s13059-021-02424-w

      


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