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    The Major Discoveries in the Mystery of How DNA Regulates Replication

    The study of deoxyribonucleic acid, otherwise known as DNA, has been an essential part of human scientific advancement, leading to discoveries of genetic causes of disease as well as the development of medicinal drugs and diagnostics.

    Furthermore, the study of DNA has enabled scientists to sequence genomes and detect potentially harmful viruses and bacteria.

    However, despite the decades of research into DNA and molecular genetics, scientists have been unable to work out exactly how DNA undergoes one of its most miraculous stages: replication. DNA replication can be seen and, until recently, how it was determined and regulated within DNA was unknown.

    Now, following a recent line of breakthroughs, we better understand how DNA manages its replication. But why did such a question arise, and how is this incredible feat achieved?

    DNA replication taking place

    Single-cell replication has been known to scientists for quite some time now but, due to studies attempting to thwart the important research being done on the replication process, DNA replication was mostly glossed over as a grand occurrence from which the commencement and conclusion were the only necessary focal points.

    Many genealogists saw this as a challenge, bent on uncovering the secrets behind this seemingly random series of events.

    Back in 2002, Alberts, Johnson, and Lewis et al. published Molecular Biology of the Cell: 4th Edition, in which they detail the process of DNA replication.

    They explain that DNA replication can only occur with the help of many different proteins:

    • DNA polymerase and DNA primase come in for the catalyzing process of nucleoside triphosphate polymerization.
    • DNA tangling issues are relieved by the protein DNA topoisomerases.
    • The DNA helix is opened up, ready for replication, through DNA-binding proteins and the DNA helicases.
    • An enzyme that degrades RNA primers and DNA ligase combine to bind discontinuously synthesized lagging-strand DNA fragments.

    In this same paper, it was surmised that many of the proteins used to facilitate DNA replication would associate with each other at the replication fork – where DNA replication takes place – to then form a highly efficient “replication machine,” according to the report.

    As the machine, they would coordinate the movements and activities of each component involved in the DNA replication.

    However, once researchers were able to record close-up footage of a single DNA molecule, that perception quickly changed.

    A rethink on how DNA replicates

    In June 2017, scientists were finally able to record footage of one DNA molecule replicating in a close-up view.

    It was the first time that the event had ever been recorded and, after much due analysis of the footage, the researchers decided that former assumptions about the replication process being organized and regimented were most likely incorrect.

    The footage, shot and shown in real-time, showed that DNA replication, this core function in living creatures, was remarkably random.

    Due to the instance of genetic mutations, it was assumed that it was the replication process randomly going wrong, which would result in mutations.

    In the footage, it was noted that there simply wasn’t any coordination between the strands, with each acting almost autonomously from the other, and yet, in spite of these independent actions, there was a perfect match at the end of the replication every time.

    What made the viewers perceive the strands as acting independently was the number of stop-starts, looking as though each strand was working to its own timeline.

    As if for no reason at all, a strand that was behind the other strand in the replication process could grind to a halt, while the other continued to synthesize and then, at other times, one could shoot up in speed.

    The lack of coordination was also seen as a reason behind the double helix of DNA being able to suddenly stop unzipping for further replication, with what they labeled a ‘dead man’s switch,’ to enable polymerase to catch-up with the process.

    As the event clearly showed a great deal of randomness, people started to rethink DNA replication and how it takes place without mutations occurring as a result.

    At the time, team member Stephen Kowalczykowski of the University of California stated that the findings “[undermine] a great deal of what’s in the textbooks.” 

    This revelation of randomness in the replication of DNA inspired geneticists to look deeper to try to uncover any potential structure behind the chaos, and identify if any elements could disrupt or trigger the process in ways previously unseen.

    Uncovering the mystery of DNA replication

    In a study published towards the end of 2018, Sima, Chakraborty, and Dileep et al. solved one of the great mysteries of DNA replication.

    The paper, Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication, explains how the team worked out that certain elements do orchestrate replication timing in DNA, compartmentalization, topologically associating domain, and transcription.

    To unlock the secrets behind the chemical architecture that orchestrates the timings behind DNA replication, the team utilized CRISPR (clustered regularly interspaced short palindromic repeats) technology in order to take out chromosomes to see what would influence the process and timings.

    CRISPR is used to identify threatening genes, such as those of viruses and, once a specific gene code is found, the enzymes of CRISPR go to break the sequence. The team used this functionality to cut out specific parts of the DNA sequence to see its impact on DNA replication.

    Initially, CRISPR was used to cut out various structures within DNA architecture, the DNA here being that of a mouse embryonic stem cell, or to switch structures around. CTCF, the CCTC-binding factor proteins binding sites, were the focus early on as the protein helps to control transcription.

    As such, its binding sites for the protein should be what governs DNA’s spatiotemporal operations. Instead, the cutting and displacement of these sequences did very little to impact the timing of DNA replication.

    After these attempts failed to yield the desired results, the team turned to a three-dimensional analysis of high-resolution imagery of the contact sites that the DNA was making with itself.

    Performing this analysis allowed the researchers to hone in on which areas were at work during the process, leading to the identification of many important areas outside of the formerly focussed-upon CTFC and its associated boundaries.

    This discovery was integral to unlocking the mystery of DNA replication timing.

    Deleting three intra-TAD (topologically associating domain) CTCF-independent 3D contact sites, resulted in a complete replication timing shift.

    It was complete chaos after the removal of these structures during the ensuing DNA replication process.

    Transcription was missed, the timing was thrown off, and the DNA architecture was made weaker as a result. It affected timing throughout the entire process, from the beginning to the end.

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    Further findings in the process of DNA replication

    Having found a controlling factor behind the timing of DNA replication, one of the many follow-up inquiries is into the selection of which origins undergo replication.

    Origins regulate the entire genome replication process, with each origin marking DNA replication starting points along the genome.

    However, of these tens of thousands of origins, only ten percent, or so, are used in each cell. Given the regulation of the individual DNA replication process, asking what governs which replication process is a natural follow-up question.

    As discovered by Dr. Guohong and Dr. Mingzhao of the Institute of Biophysics of the Chinese Academy of Sciences, it is H2A.Z – a histone variant – which facilitates the selection of DNA replication origins as well as their activation.

    During the study, which was published in December 2019 – almost a year on from the finding of Sima, Chakraborty, and Dileep et al. – mass spectrometry revealed that many of the subunits involved in the pre-replication complex were enriched by H2A.Z, suggesting that the mono-nucleosomes may play a part in the selection of DNA replication origins.

    Building from that observation, the research went on to reveal that the H2A.Z-regulated DNA replication origins did, indeed, have a higher firing efficiency.

    It was also found that the origins regulated by H2A.Z would replicate earlier than other origins not regulated by the histone variant.

    The study into eukaryotes – organisms with cells that have the nucleus within a membrane (animals, humans, plants) – gives an understanding of the mechanisms at work when DNA replication origins are selected.

    The potential of further understanding DNA replication regulation

    Understanding our DNA and how our DNA works can lead to tremendous steps forward in medical sciences.

    Just as understanding your own DNA and lineage through the best DNA testing kits can help you to find out if you’re predisposed to any medical conditions, uncovering how DNA replicates can allow scientists and doctors to alter the process to suit their goals.

    The study, published in 2018, concerning the timing of DNA replication, could open up more avenues of research into health and pathology; namely identifying the processes which result in certain diseases. The most recent 2019 study into DNA replication origin activation could act as a pathway to targeting cancer treatment as well as regulating T-cell functions during immunotherapy.

    Further study into the mechanics and processes of our DNA could lead to incredible medical revelations, with the two detailed here already showing the potential of leading to tremendous breakthroughs in treatment and prevention.