Residues within the N-terminal Domain of Human Topoisomerase I Play a Direct Role in Relaxation*

Abstract

All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, Eukaryotic topoisomerase I (topo I)1 is a monomeric enzyme that plays a major role in important cellular processes by regulating the topology of DNA. The enzyme relaxes negative and positive supercoils arising as a consequence of DNA processes such as DNA transcription, replication, recombination, and chromosome condensation (1). Mechanistically, topo I acts by introducing transient singlestrand breaks into the DNA double helix. The catalytic cycle can be subdivided into several steps including: (i) non-covalent DNA binding, (ii) cleavage, (iii) strand rotation, (iv) religation, and (v) enzyme turnover. The cleavage and religation events constitute two reverse phosphoryl transfer (transesterification) reactions. During the cleavage reaction, an active-site tyrosine residue of the enzyme is used as a nucleophile to break a phosphodiester bond of the DNA backbone, generating a covalent enzyme-(3Ј-phosphotyrosyl)-DNA linkage and a free 5Ј-hydroxyl group (2-4). This 5Ј-hydroxyl group provides the nucleophile for the religation reaction that restores intact DNA.The solved crystal structure of an N-terminal-truncated version of the human topo I together with proteolytic analyses show that the enzyme is organized into four structural domains. These consist of an N-terminal domain (amino acids 1-206), a core domain (amino acids 207-635), a linker domain (amino acids 636 -712), and a C-terminal domain (amino acids 713-765) (2, 3). The C-terminal domain contains the active-site tyrosine (Tyr 723 ), which together with the catalytic residues Arg 488 , Lys 532 , Arg 590 , and His 632 of the core domain constitutes the active site of the enzyme (4 -8). Structural data show that the core and C-terminal domains form a clamp structure that wraps around the DNA and, together with the helix-turnhelix linker domain (1), contacts DNA in a region extending 4 base pairs upstream and 9 base pairs downstream of the cleavage site (3). Based on this structural information of the human topo I-DNA complex, a model for strand rotation (topoisomerization) has been proposed. According to this "controlled rotation" model, rotation of the cleaved strand around the intact strand is partially hindered by contacts between the rotating DNA and part of the core and linker domains (3). The involvement of the linker ...

SF2/ASF protein inhibits camptothecin‐induced DNA cleavage by human topoisomerase I

“…This region is included in the N-terminal domain of the enzyme that in general is dispensable for both relaxation and binding of camptothecin [14,15]. However, detailed studies have recently revealed that the N-terminal domain influences the rate of relaxation and sensitivity of the enzyme to camptothecin [16].In the present work we report that the natural substrate for the kinase activity of htopo I, SF2/ASF protein, inhibits camptothecin-induced DNA cleavage by the enzyme. This effect results from reduced amount of the cleavable complex formed by htopo I in the presence of SF2/ASF.…”

“…Elimination of a dissociation of htopo I as a rate-limiting step for relaxation can be achieved by decreasing the DNA/htopo I ratio during the relaxation to 1 : 1 or less [16]. However, in this case the reaction rate should be slowed because of an elevated amount of the enzyme [16]. We achieved this by reducing the reaction temperature to 0°C.…”

“…On the other hand, an impaired DNA cleavage observed in the cleavage assay should be reflected by inhibition of the reaction catalysed by htopo I rather than by inhibition of the movement of the enzyme between DNA substrate molecules. Elimination of a dissociation of htopo I as a rate-limiting step for relaxation can be achieved by decreasing the DNA/htopo I ratio during the relaxation to 1 : 1 or less [16]. However, in this case the reaction rate should be slowed because of an elevated amount of the enzyme [16].…”

“…This region is included in the N-terminal domain of the enzyme that in general is dispensable for both relaxation and binding of camptothecin [14,15]. However, detailed studies have recently revealed that the N-terminal domain influences the rate of relaxation and sensitivity of the enzyme to camptothecin [16].…”

The N-terminal Domain Anchors Human Topoisomerase I at Fibrillar Centers of Nucleoli and Nucleolar Organizer Regions of Mitotic Chromosomes

“…This domain distinguishes eukaryotic topoisomerase I from a minimal variant encoded by vaccinia virus (24) and from other microbial recombinases more distantly related (25). It contributes scarcely to the activity of the enzyme in vitro (26), but it is believed to determine the biological properties of topoisomerase I. Most notably, it seems to be a docking place for interacting proteins, such as nucleolin (27).…”