International Business Department           Liu Bojia           March 26, 2025

  DNA replication is a key step in cell division and an essential process for the survival and reproduction of living organisms. We all know that DNA has two complementary strands, and it exists in the form of a double helix, with various chemical bonds responsible for maintaining a stable helical structure. Therefore, the first step in the replication of DNA is to unravel the helical structure so that the double-stranded DNA becomes single-stranded DNA.

  Scientists have long since discovered that this heavy lifting is done primarily by the enzyme hexameric deconjugase. In the process of DNA replication, deconjugating enzyme can quickly unravel double-stranded DNA to provide single-stranded templates for replicative enzymes to ensure the accurate transmission of genetic information; in the process of DNA recombination, deconjugating enzyme will help DNA molecules to carry out structural reorganisation to promote the diversity and adaptability of genes. However, this seemingly mechanical process hides many unanswered questions, such as how does the deconjugating enzyme choose its entry point and how does ATP help the deconjugation process?

  A recent study in Nature has successfully answered these questions. With the help of cryo-electron microscopy (cryo-EM) technique, the researchers revealed the structural dynamics of simian virus 40 (SV40) LTag deconjugase during DNA replication. The study reveals at the atomic level the interactions between DNA, deconjugase, and ATP, and how all three synergistically contribute to DNA helix unwinding. This is the most detailed description of the first step of DNA replication ever given by scientists, and represents an entirely new milestone in the study of deconjugases.

  In this study, the researchers first used cryo-electron microscopy to capture multiple conformational states of LTag deconjugase during DNA replication. They then successfully resolved the conformational landscape of LTag binding to forked DNA under catalytic conditions and the coordinated movement of DNA displacement and deconvolution by continuous heterogeneity analysis.

  Firstly, LTag consists of two layers (N-layer and C-layer) that form a symmetric hexameric ring structure; the N-layer is responsible for stabilising the whole molecule, while the ATP-active region in the C-layer is the core engine that drives DNA movement. At the start of replication, the LTag assembles as a ‘head-to-head’ dimer, simultaneously cutting the DNA at two symmetric sites to form a bidirectional replication fork. Each hexamer of the enzyme grips single-stranded DNA through six DNA-binding loops.

  After the DNA-binding ring is inserted into the DNA double strand, LTag breaks the hydrogen bonds between the DNA double strands. Typically, the replication start point is chosen to be in a region rich in A-T base pairs. These regions have weak inter-base forces and are ideal starting points for replication initiation.

  More notably, the study identified a special ATP-driven mode, the entropy switch, which achieves deconvolution by removing shift barriers on the DNA strand, rather than directly driving DNA movement. This also reverses the conventional wisdom that ATP hydrolysis directly powers DNA unwinding.

  Specifically, when ATP hydrolysis occurs, the interface between the subunits at the top of the deconjugate enzyme shifts from ATP-type to ADP-type, which results in the rotation of the C-layer relative to the N-layer, thereby driving DNA along the deconjugate channel. This process is analogous to a ‘ratchet’ mechanism that allows the DNA to progressively pass through the deconjugase enzyme and be deconvoluted. Since this mechanism releases stored elastic potential energy by changing the ordering of the protein conformation, it can help to reduce wasted energy and increase the catalytic efficiency of the deconjugase enzyme.

  Professor Alfredo De Biasio, corresponding author of the study, said: ‘From a design point of view, deconjugate enzymes are a good example of energy-efficient mechanical systems. Such nanomachines designed using entropy-change switches can perform driving tasks with energy-efficient mechanisms.’

  The new study not only reveals the structure and function of deconjugating enzymes at the molecular level, but also provides important information for understanding the first step of DNA replication. In addition, this study also found that similar working mechanisms exist for viral E1 deconjugase and bacterial DnaB deconjugase, which also provides an important theoretical basis for the development of new antiviral and antibacterial drugs.