Chongfan Technology
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05
2024
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08
NAR | Jennei team reveals the role of Tower domain, which is involved in anchoring and releasing TOPRIM domain, thus triggering Top2 to bind and cleave DNA.
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The ability to catalyze reversible DNA cleavage and recombination is central to the role of topoisomerases in regulating DNA topology.In type IIA topoisomerases (Top2), the formation of DNA cleavage centers is driven by structural rearrangements induced by DNA binding. These changes place key catalytic modules, such as the active site tyrosine of the WHD domain and the metal ion chelated by the TOPRIM domain, around a cleavable phosphodiester bond for reversible transesterification.
On July 30, 2024, Zhan Yuli, National Taiwan University of China, as the corresponding authorNucleic Acids Research Published online entitled“Structural insights into the assembly of type IIA topoisomerase DNA cleavage-religation center”research paper, the studyA previously unrecognized role for the Tower domain was revealed, highlighting its involvement in anchoring and releasing the TOPRIM domain, which triggers Top2 for DNA binding and cleavage.
In order to understand the assembly process in detail, the catalytic core structure of human Top2α and Top2β in the pathway conformation state was expounded. This state is characterized by a switch at the interface between the Tower and the opposite TOPRIM domain, exposing a groove for accommodating incoming G-segment DNA. Structural overlay further reveals how subsequent DNA binding-induced detachment of the TOPRIM and Tower domains allows for a firm grasp of bound DNA cleavage/religion. It is worth noting that,Studies have identified a previously undocumented protein-DNA interaction between the arginine end of the α-helix of the TOPRIM domain and the DNA backbone, which is important for Top2 function.
DNA topoisomerases (Tops) are ubiquitous enzymes that are essential to all three areas of life.These enzymes solve the topological problems arising from cellular DNA trading, such as replication, transcription, repair, and chromosome segregation. A common feature of all types of Top is that they can reversibly cut and recombine the DNA backbone, thereby being able to manipulate DNA topology without compromising the integrity of genome structure.According to whether they cut single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA),Tops are divided into type I and type II, respectively.
Each type is further divided into subtypes based on sequence differentiation, structural features, and mechanistic differences. Members of type IB and type IC subfamilies effectively release DNA supercoiled stress by introducing a gap in one DNA strand, allowing controlled rotation of the gap strand around the uncut strand. In contrast, type IA and type II enzymes change DNA topology through the so-called "chain channel mechanism", where they briefly break and introduce an opening (or gate) in ssDNA or dsDNA (called gate or G segment DNA), respectively, and subsequently transport another DNA strand or double strand (called transfer or T segment DNA) through the gate.Since reversible DNA cleavage is central to all Tops, a thorough understanding of their catalytic mechanism requires a detailed understanding of this process.
Eukaryotic Top2 is mainly a homodimer, assembled from two identical multidomain subunits, each of which is usually between 1400 and 1600 residues, mainly due to the C- terminal domain (CTD). Species and isoform-specific variation.Starting from the N-terminal, a single subunit includes the following domains: GHKL ATPase domain, transducer domain, topoisomerase primer enzyme (TOPRIM) domain, wing helix domain, tower domain, coiled coil region and Primary dimerization domain, and ends with an essentially disordered and less conservative CTD.
Top2 has an overall structure of 2-fold symmetry, containing a DNA cleavage-ligation center consisting of two TOPRIM domains, two WHD domains, and two Tower domains, suitable for introducing double-stranded DNA breaks (DSBs). The TOPRIM domain contains a catalytic Mg2 Bound metal-binding DxD motifs and two conserved DNA-binding elements, EGDSA and PLRGK motifs. The WHD domain, with a winged helix-turn-helix motif and various conserved and surface-exposed residues, cooperates with the TOPRIM and Tower domains to establish a DNA-binding groove that binds to the G-segment DNA.Once bound, the formation of extensive non-covalent interactions induces bending of the G-segment DNA and drives its transition to the-type conformation within the bound region.
Graphical Summary
In addition, the WHD domain contains the active site tyrosine residue, as opposed to the TOPRIM domain of the subunit providing Mg2 together catalyze a reversible transesterification reaction.Transient DSB formation is achieved by two symmetrically arranged active site tyrosine residues attacking a pair of phosphodiester bonds staggered by 4 base pairs on opposite strands. This protein-linked DSB unlocks the DNA backbone and provides a portal, the DNA portal called Top2, through which another DNA duplex can be transported to regulate DNA topology. During DNA passaging, Top2 undergoes extensive conformational changes, including coordinated formation and subsequent disruption of connections between different domains of the enzyme.This process is characterized by highly ordered structural reordering, which enables the directional transport of T-segment DNA through broken G-segments, thus achieving changes in DNA topology.
The study elaborated that human Top2 alpha (hTop2 alphacore) and Top2β (hTop2βcore) of the DBCC crystal structure is in a conformational state capable of binding DNA.This state is characterized by the anti-assembly of two symmetry-related interfaces, each formed between the Tower domain of one subunit and the TOPRIM domain of the other subunit. Such an arrangement results in the formation of extensive DNA binding slots, optimally configured to accommodate incoming DNA duplexes. Structural overlay further revealed subsequent Top2 conformational changes induced by G-segment DNA, including detachment of the TOPRIM domain from Tower and repositioning toward bound DNA, leaving the bound double strand tightly held and ready for cleavage and repair. It is worth noting that the study found an unprecedented protein-DNA interaction. In the helix bundle region where the function of TOPRIM domain is not recognized, the C- terminus of the α-helix is bridged with the backbone of the G segment through a strictly conserved arginine residue, which plays an important role in the function of Top2.The study revealed how the Tower-TOPRIM interface formed in trans initiates the G-segment binding enzyme, and how the DNA binding-induced disruption of this interface initiates the formation of the DNA cleavage/ligation center of Top2.
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