Sumoylation of Topoisomerase I Is Involved in Its Partitioning between Nucleoli and Nucleoplasm and Its Clearing from Nucleoli in Response to Camptothecin

Abstract

Previous studies identified a small fraction of putatively sumoylated topoisomerase I (TOP1) under basal conditions (ϳ1%), and anticancer camptothecins that trap the TOP1-DNA covalent intermediate markedly increase the sumoylation of TOP1 (<10%). To study the role of the sumoylation of TOP1, we mutated sites on green fluorescent protein (GFP)-TOP1 corresponding to the consensus sequence for protein sumoylation (⌿KXE, where ⌿ is a hydrophobic residue) and assayed the mutants for basal and camptothecin-induced sumoylation. Only one of the eight mutants, K117R, located in the highly charged NH 2 -terminal region, showed a substantial reduction (ϳ5-fold) in basal and camptothecin-induced sumoylation; thus, Lys-117 appears to be the major sumoylation site. A triple mutant having the ⌿KXE sequences flanking K117R additionally mutated (K103R/ K117R/K153R) showed little if any sumoylation, but was degraded like wild-type GFP-TOP1 during camptothecin treatment. However, K103R/K117R/K153R-GFP-TOP1 was markedly concentrated within nucleoli, depleted from the remainder of nucleus, and failed to be cleared from nucleoli in response to camptothecin treatment. These data are consistent with a model wherein basal transient sumoylation of the NH 2 -terminal, highly charged, disordered region prevents TOP1 binding to sites in nucleoli, thus driving it to bind in the nucleoplasm; and camptothecin treatment, which increases TOP1 sumoylation, further shifts the binding resulting in delocalization of TOP1 from nucleoli to nucleoplasm.

SUMO: a regulator of gene expression and genome integrity

“…These agents block the catalytic cycle of DNA breakage and religation and ultimately induce DSBs during DNA replication. Remarkably, topoisomerase inhibitors strongly stimulate SUMOylation of topoisomerase I (Horie et al, 2002;Mao et al, 2000b) and SUMOylation seems to regulate its partitioning between nucleoli and nucleoplasm, since a SUMO-deficient mutant of topoisomerase I is trapped in the nucleolus after camptothecin treatment Rallabhandi et al, 2002). This finding substantiates the view that SUMOylation addresses proteins to specific nucleoplasmic sites in response to DNA damage.…”

Distinct Effects of Topoisomerase I and RNA Polymerase I Inhibitors Suggest a Dual Mechanism of Nucleolar/Nucleoplasmic Partitioning of Topoisomerase I

“…1C indicate catalytic activity as the sole determinant of nucleolar depletion of topo I in response to camptothecin, which is in clear contrast to previous observations by others suggesting sumoylation of lysine residues 103, 117, and 153 to play a crucial role in this process (14). This contradiction could be due to the fact that we have investigated here huge truncations of topo I, whereas the conflicting data were obtained by silencing just the relevant sumoylation sites in the full-length enzyme by point mutations.…”

“…Thus, partitioning of topo I between nucleoli and nucleoplasm seems in general governed by mobility gradients within the cell nucleus, with nucleolar accumulation reflecting the enzyme's lesser mobility in the nucleoli, and relocation to the nucleoplasm in response to camptothecin reflecting attenuation of the enzyme at nucleoplasmic sites, where it is actively processing genomic DNA (12). However, this simple explanation was disputed, because camptothecin also stimulates modification of topo I with small ubiquitin-like modifiers (SUMO) (13), and mutational silencing of the major target site of topo I for this modification (K103R,K117R,K153R) enhances nucleolar accumulation of the enzyme and abolishes its nucleolar clearance in response to camptothecin (14). This has been interpreted as an indication of an active and directed transport of topo I between nucleoli and nucleoplasm that is triggered by the attachment of SUMO to (sumoylation of) the enzyme.…”

Mechanisms of resistance to topoisomerase I-targeting drugs

“…Although SUMO is similar to ubiquitin in structure, SUMO does not target proteins for degradation, but is implicated in control of cellular localization (Muller et al, 2001). Indeed, sumoylation of Top1 is associated with relocalization of the protein from the nucleolus to a more diffuse nuclear pattern following CPT treatment (Mo et al, 2001), whereas, Top1 mutants that cannot be sumoylated remain more concentrated in nucleoli of cells even after CPT treatment (Rallabhandi et al, 2002). Together these studies strongly suggest that sumoylation regulates Top1 localization in the nucleus, and that sumoylation of Top1 may function to decrease Top1-DNA interactions and thus minimize Top1-mediated DNA damage induced by CPT.…”