SUMO-1 conjugation to topoisomerase I: A possible repair response to topoisomerase-mediated DNA damage

Abstract

Ubiquitin͞26S proteasome-dependent degradation of topoisomerase I (TOP1) has been suggested to be a unique repair response to TOP1-mediated DNA damage. In the current study, we show that treatment of mammalian cells or yeast cells expressing human DNA TOP1 with camptothecin (CPT) induces covalent modification of the TOP1 by SUMO-1͞Smt3p, a ubiquitin-like protein. This conclusion is based on the following observations: (i) Mammalian DNA TOP1 conjugates induced by CPT were cross-reactive with SUMO-1͞Smt3p-specific antibodies both in yeast expressing human DNA TOP1 as well as mammalian cells. (ii) The formation of TOP1 conjugates was shown to be dependent on UBC9, the E2 enzyme for SUMO-1͞Smt3p. (iii) TOP1 physically interacts with UBC9. (iv) Ubc9 mutant yeast cells expressing human DNA TOP1 was hypersensitive to CPT, suggesting that UBC9͞SUMO-1 may be involved in the repair of TOP1-mediated DNA damage.UBC9 ͉ Smt3p ͉ ubiquitin T opoisomerase-mediated DNA damage represents an unique type of DNA damage whose importance has become increasingly appreciated. Many antibiotics, anticancer drugs, toxins, carcinogens, and physiological stresses are known to abort the catalytic cycles of topoisomerases resulting in the formation of topoisomerase-mediated DNA damage (1-7). Despite its importance, little is known about the molecular basis for the repair of this unique type of DNA damage.Recent studies have demonstrated that topoisomerase I-(TOP1) mediated DNA damage activates a ubiquitin͞proteasome pathway, resulting in degradation of TOP1 (down-regulation of TOP1) (8). It has been speculated that this pathway may represent a unique repair mechanism for TOP1-mediated DNA damage because down-regulation of human TOP1 (hTOP1) is expected to result in resistance͞tolerance to TOP1 poisons (8).Human SUMO-1 (small ubiquitin-like modifier) is an 11-kDa protein that shares 18% sequence homology with ubiquitin (9-11). Smt3p, which is 48% identical to human SUMO-1, has been identified in yeast (12,13). SUMO-1͞Smt3p is activated by a heterodimeric E1 enzyme in both the yeast and mammalian systems (13)(14)(15). In addition, UBC9, an E2 enzyme, has been shown to specifically conjugate SUMO-1͞Smt3p to the target proteins (11,(16)(17)(18). Recently, an enzyme Ulp1p, which specifically cleaves proteins from SUMO-1͞Smt3p conjugates and is distinct from isopeptidases for ubiquitin conjugates, also has been identified in yeast (19). These results suggest that the SUMO-1͞Smt3p pathway is very similar to the ubiquitin pathway but distinct E1 and E2 enzymes as well as proteases are involved in these pathways. The biological function of the SUMO-1͞Smt3p pathway appears diverse. UBC9 was originally identified to play an essential function in G 2 -M cell cycle progression in yeast (20). However, UBC9 is also known to be important for DNA repair (21). It has been shown that SUMO-1 can be conjugated to a number of proteins such as PML (22), RanGAP1 (23, 24), I B␣ (25), FAS͞apolipoprotein-1 (26), p53 (27, 28), RAD51, and RAD52 (29). A growing number of pr...

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

“…These results suggest that Lys-117 is the major sumoylation site on TOP1, and that the sumoylation of the sites flanking Lys-117 is sufficient to enable TOP1 to be cleared from nucleoli in response to CPT and to prevent the accumulation of TOP1 in nucleoli under basal conditions. Mutation of only a single lysine (Lys-117) eliminated the majority of sumoylated TOP1 despite the fact that up to six SUMO molecules have been seen attached to TOP1 (2,20). These observations can be explained by more than one model.…”

“…Currently, the multiple monosumoylation model appears to be most consistent with our experimental data and with the fact that single SUMO conjugates on four closely spaced (IVL)KXE sites have been mapped on Cdc3 (25). Notably, using antibodies specific to SUMO-1 or SUMO-2/3, we and others have observed SUMO-2/3 and SUMO-1 conjugated to TOP1 (20,21). 3 The close juxtaposition of multiple sumoylation sites on TOP1 (Lys-103, Lys-117, Lys-153) is similar to that found on the Saccharomyces cerevisiae septins Cdc3 (Lys-4, Lys-11 , Lys-30, Lys-63) and Shs/Sep7 (Lys-426, Lys-437) (25), the cytomegalovirus protein IE2 (Lys-175, Lys-180) (26), HDAC1 (Lys-444, Lys-476) (27), and GRIP1 (Lys-731, Lys-788) (28) (see Table I).…”

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

“…Nucleolar Targeting of Topoisomerase I Does Not Depend on the Presence of the Major Sumoylation Site-It has been shown that camptothecin stimulates modification of topo I with ubiquitin (18) and SUMO (13) and that these modifications play a crucial role in resistance of tumor cells to camptothecin, because they trigger proteolytic degradation of DNA-linked topo I (40). Thus, it is clear that both effects are important cellular responses to topo I-mediated DNA damage.…”

“…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.…”

RECQ5-dependent SUMOylation of DNA topoisomerase I prevents transcription-associated genome instability

“…SUMOplot analysis predicted that human TOP1 contains 12 motifs that have high probabilities for SUMOylation, and TOP1 has been shown to be poly-SUMOylated at K103, K117 and K153 in response to CPT [23][24][25][26] . However, we found that transcriptionassociated TOP1 SUMOylation is distinct from the SUMOylation events induced by CPT.…”