Structural and biochemical basis for DNA and RNA catalysis by human Topoisomerase 3β

Abstract

In metazoans, topoisomerase 3β (TOP3B) regulates R-loop dynamics and mRNA translation, which are critical for genome stability, neurodevelopment and normal aging. As a Type IA topoisomerase, TOP3B acts by general acid-base catalysis to break and rejoin single-stranded DNA. Passage of a second DNA strand through the transient break permits dissipation of hypernegative DNA supercoiling and catenation/knotting. Additionally, hsTOP3B was recently demonstrated as the human RNA topoisomerase, required for normal neurodevelopment and proposed to be a potential anti-viral target upon RNA virus infection. Here we elucidate the biochemical mechanisms of human TOP3B. We delineate the roles of divalent metal ions, and of a conserved Lysine residue (K10) in the differential catalysis of DNA and RNA. We also demonstrate that three regulatory factors fine-tune the catalytic performance of TOP3B: the TOP3B C-terminal tail, its protein partner TDRD3, and the sequence of its DNA/RNA substrates.

What’s on the Other Side of the Gate: A Structural Perspective on DNA Gate Opening of Type IA and IIA DNA Topoisomerases

“…The binding of ssDNA (G-segment) to the DNA-binding groove, which is running along the domain DIV, induces a domain rearrangement, which leads to the formation of the catalytically competent active site and aligns the scissile phosphodiester bond for cleavage [ 60 , 61 , 62 ] ( Figure 3 b, states 1 and 2). Although the G-strand is held in the DNA binding groove mostly via interactions with the DNA backbone [ 62 , 63 , 64 ], type IA topoisomerases usually display DNA sequence selectivity for cleavage [ 65 , 66 ]. A cytosine residue at position −four relatives to the cleavage site is required for the bacterial topoisomerase IA enzymes.…”

“…A crystal structure of E. coli topoisomerase I, a covalent intermediate, revealed that the enzyme has a cavity that specifically accommodates this base [ 68 ] and acts as a molecular ruler that helps to precisely position the scissile bond in the active site [ 65 ]. Interestingly, it was shown that for human topoisomerase IIIβ, the sequence requirements for RNA cleavage significantly differ from the DNA cleavage sequence [ 66 ]. To date, there is no structure of type IA topoisomerase bound to RNA that would explain the molecular basis for the differential selectivity.…”

“…The eukaryotic Topo III enzymes additionally have RGG domains for RNA-binding [ 94 ]. These domains on the C-terminus bind DNA, usually with high affinity [ 62 , 95 ], forming additional stabilising interactions with the substrate that enhance the activity of the core enzyme [ 66 , 96 ]. Though the exact role of CTD is unknown, it is essential for catalytic activity in some topoisomerases but not in others, and its deletion only impairs enzyme processivity [ 59 , 66 , 86 , 96 , 97 , 98 , 99 ].…”

“…These domains on the C-terminus bind DNA, usually with high affinity [ 62 , 95 ], forming additional stabilising interactions with the substrate that enhance the activity of the core enzyme [ 66 , 96 ]. Though the exact role of CTD is unknown, it is essential for catalytic activity in some topoisomerases but not in others, and its deletion only impairs enzyme processivity [ 59 , 66 , 86 , 96 , 97 , 98 , 99 ]. For example, E. coli and mycobacterial TopI absolutely require the CTD for their DNA relaxation activity, and it was proposed that the CTD binds the T-strand to promote its passage [ 62 , 85 ].…”

“…TDRD3 also binds to domain DII of TopIIIβ at the same position, interacting with the hinges and possibly regulating gate opening ( Figure 4 ). Yang et al demonstrated that TDRD3 enhances DNA relaxation by TopIIIβ and proposed that their interaction could reduce the flexibility of the DNA-gate to facilitate T-strand passage and/or G-strand re-ligation [ 66 ].…”