Targeting to Transcriptionally Active Loci by the Hydrophilic N-Terminal Domain of Drosophila DNA Topoisomerase I

Abstract

DNA topoisomerase I (topo I) from Drosophila melanogaster contains a nonconserved, hydrophilic N-terminal domain of about 430 residues upstream of the conserved core domains. Deletion of this N terminus did not affect the catalytic activity of topo I, while further removal of sequences into the conserved regions inactivated its enzymatic activity. We have investigated the cellular function of the Drosophila topo I N-terminal domain with top1-lacZ transgenes. There was at least one putative nuclear localization signal within the first 315 residues of the N-terminal domain that allows efficient import of the large chimeric proteins into Drosophila nuclei. The top1-lacZ fusion proteins colocalized with RNA polymerase II (pol II) at developmental puffs on the polytene chromosomes. Either topo I or the top1-lacZ fusion protein was colocalized with RNA pol II in some but not all of the nonpuff, interband loci. However, the fusion proteins as well as RNA pol II were recruited to heat shock puffs during heat treatment, and they returned to the developmental puffs after recovery from heat shock. By immunoprecipitation, we showed that two of the largest subunits of RNA pol II coprecipitated with the N-terminal 315-residue fusion protein by using antibodies against ␤-galactosidase. These data suggest that the topo I fusion protein can be localized to the transcriptional complex on chromatin and that the N-terminal 315 residues were sufficient to respond to cellular processes, especially during the reprogramming of gene expression.Type I DNA topoisomerase (topo I) catalyzes a cycle of transesterification reactions, during which it generates transient single-strand DNA breaks; these reversible reactions result in the topological transformation of either single-strand or double-strand DNA molecules (8,20,55). Biochemical and genetic studies have demonstrated that topo I plays important roles in DNA replication, transcription, and recombination and in chromosome condensation and the maintenance of genome stability. The top1 gene is not essential for viability in yeast, and genetic analysis suggests that some of the biological functions of topo I can be performed by topo II (59). However, recent genetic screens of Saccharomyces cerevisiae have identified four complementation groups of mutants with mutations that, in combination with a top1 mutation, result in a lethal phenotype. Some of these synthetic lethal mutants have mutations in genes other than top2 (44). Therefore, it is possible that not all the biological functions of topo I can be substituted by topo II. Furthermore, because top1 is an essential gene in Drosophila melanogaster (30) and in mice (37), the functions of topo I may not be efficiently substituted by topo II in multicellular organisms.One of the functions of topo I is to provide a swivel to facilitate the unwinding of the DNA duplex during the process of transcription or replication (56). Diametric DNA supercoiling can transiently accumulate in the front and in the wake of a transcriptional fork as proposed...

Disruption of a DNA Topoisomerase I Gene Affects Morphogenesis in Arabidopsis

“…Topo I has been shown to be involved actively in the transcription process in animal systems (Merino et al, 1993;Shykind et al, 1997;Shaiu and Hsieh, 1998). Furthermore, the top1␣-1 mutant phenotype also seems to be in line with recent genetic data that have highlighted the importance of chromatin remodeling and assembly in the control of plant morphogenesis (Verbsky and Richards, 2001).…”

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

“…The other is that it is heavily involved there in the relaxation of super-coils generated during transcription of rDNA. The second model is supported by the finding that the N-terminal domain targets topoisomerase I to sites of transcriptional activity (34) and by the observation that ongoing transcription seems to recruit topoisomerase I to the nucleoli (12). The inverse should be the case, if the enzyme was stored there in an inactive form.…”

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

“…Second, it contains the nucleolin binding site (32), and in Saccharomyces, the nucleolin ortholog, Nsr1p, physically interacts with yeast TOP1; and null mutants of NSR1 exhibited altered localization of TOP1 (33). Third, the NH 2 terminus of Drosophila TOP1 was sufficient for recruitment to active sites of transcription (34). Finally, the NH 2 -terminal region per se fused to GFP has been shown to concentrate in nucleoli and to clear from nucleoli in response to CPT (7,35) or ultraviolet light (35), which indicates that formation of the covalent TOP1-DNA intermediate is not required for nucleolar clearing.…”