User:Sabrina K.K. Komatsu/Sandbox 1

DNA Topoisomerases (TOP) are enzymes capable of solving topological problems in DNA molecules during replication, transcription, recombination and chromatin remodeling by introducing temporary breaks in the DNA. Besides that, these enzymes can modulate the level of DNA supercoiling to facilitate protein interaction with the molecule and to prevent supercoiling relationed stress. There are several types of topoisomerases that are different in structure and function, but basically, the topos are divided based on whether they catalyze single- (type I) or double-stranded (type II) DNA breaks.

Within type two topoisomerases there are two classes: A and B. Class B is found only in archaea and type A is present in eukarya and bacteria. Humans have two types of 2A topoisomerases: isoforms alpha and beta. Topoisomerase 2 alpha has a larger role in chromosome segregation and DNA replication and tends to be expressed in more proliferating tissues, which makes topoisomerase 2 alpha a cancer cell marker and the target of anti-cancer drugs. Top 2A alpha is responsible for the selective cleaving, rearranging and religation of DNA strands, which helps untangle the chromosome, thereby changing the state of DNA in the cell. Mammalian DNA topoisomerase IIα (human) IIA is a homodimeric protein, in whitch each subunit structure can be broken down into three major components that are connected by hinged like regions: the N gate, the DNA gate and the C gate.

Function

During replication, transcription and cell division, DNA passes for various states of coiling, so sometimes DNA can become too coiled, which, without alleviation, can cause DNA brakes or arrest of the replication, cell divisior or transcription. To prevent breakages from happening, top 2a cuts both strands of the DNA duplex, passes another DNA duplex through the cleaved one and religate the cut duplex to release tension. This is a risky task, once with not completed properly, it damages the state of the crhomatin and can result in cell death.

In addition to the better-known function of undoing super-coilings in DNA, TOP2a was shown to be important for comosomal condensation and maintenance of chromosomal structure, since chromatic compaction appears to arise, in part, due to the interaction between topo 2a and complexes of structural maintenance of chromosomes (SMC complexes).Topo IIa is also involved in chromosome segregation during anaphase and in chromatid resolution at ribosomal DNA (rDNA).

Structure

Mammalian DNA topoisomerase IIα (human) IIA is a homodimeric protein, in whitch each subunit structure can be broken down into three major components that are connected by hinged like regions: the N gate, the DNA gate and the C gate. The DNA topoisomerase IIα are represented wuth two monomeric structures, one in green and one in gray. This protein is bonded to .

Within the DNA gate there are three important domains: the topoisomerase primases (toprim domain), the winged helix domain (WHD domain or 5Y-CAP)) and the tower domain. The TOPRIM domain contains a DxD motif, where metal ion binding occurs due to two aspartates at positions 541 and 543, which coordinates a single magnesium 2 plus ion quelation,and a glutamate residue, that donates a proton to the sugar hydroxyl of the DNA during cleavage and abstracting the proton from the 3ʹ-OH during re-ligation. The TOPRIM domain also contributs to DNA binding, due to conserved residues, namely the EGDS and PLRGK motifs, which interact with the G-segment.The WHD contains a helix-turn-helix fold, common in DNA-binding proteins, contains catalytic tyrosine residues, responsable for forming a reversible covalent bond with the 5ʹ-scissile DNA phosphate. Besides that, The WHD also holds an isoleucine, which intercalates into the G-segment (the first segment of DNA duplex that enter the enzyme) producing a ∼150° bend,promoting DNA cleavage. So, the cleaving of the DNA backbone occurs in a bipartite active site formed by the TOPRIM DxD motif and the active site tyrosine of the WHD.

Operation

Top2A starts out with its N gates dissociated allowing it to bind a DNA duplex passing it through the N gate and binding at the DNA gate (G-segment). Binding at the DNA gate causes a significant one ~150 degree bend, promoted by an isoleucine present in the WHD domain. The N gate contains an ATPase region and ATP binding here triggers the binding of a second DNA duplex at the N gate (T-segment). Binding of ATP also causes the previously separated N gates to dimerize and close locking in the T-segment. Closing the N gates emulates conformation change that promote the cleavage of the G segment, which is catalyzed by a pair of symmetrically-related tyrosines 9,10, in conjunction with a Mg2+ ion-binding in the TOPRIM fold. Besides that, the 150 degree bend on the duplex puts bonds under significant stress, facilitating the cleavage. Once the G segment has been cleaved, it remains bound at the DNA gate, but the DNA gate opens to allow the T segment to exit through the open C gate. Than, the DNA gate closes again to religate the G segment before releasing it. Strand passage and gate openings are coordinated by defined ATPase domains12–14 which use ATP binding and hydrolysis to promote conformation changes. In more simple terms, in the type IIA reaction cycle, one double-stranded DNA is bound and cleaved by the enzyme, while a second duplex is transported through the break.

Disease

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Structural highlights

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