Activities of topoisomerase I in its complex with SRSF1.
Human DNA topoisomerase I (topo I) catalyzes DNA relaxation and phosphorylates SRSF1. Whereas the structure of topo I complexed with DNA has been resolved, the structure of topo I in the complex with SRSF1 and structural determinants of topo I activities in this complex are not known. The main obstacle to resolving the structure is a contribution of unfolded domains of topo I and SRSF1 in formation of the complex. To overcome this difficulty, we employed a three-step strategy: identifying the interaction regions, modeling the complex, and validating the model with biochemical methods. The binding sites in both topo I and SRSF1 are localized in the structured regions as well as in the unfolded domains. One observes cooperation between the binding sites in topo I but not in SRSF1. Our results indicate two features of the unfolded RS domain of SRSF1 containing phosphorylated residues that are critical for the kinase activity of topo I: its spatial arrangement relative to topo I and the organization of its sequence. The efficiency of phosphorylation of SRSF1 depends on the length and flexibility of the spacer between the two RRM domains that uniquely determine an arrangement of the RS domain relative to topo I. The spacer also influences inhibition of DNA nicking, a prerequisite for DNA relaxation. To be phosphorylated, the RS domain has to include a short sequence recognized by topo I. A lack of this sequence in the mutants of SRSF1 or its spatial inaccessibility in SRSF9 makes them inadequate as topo I/kinase substrates.
Institute of Biochemistry, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.
Takao
T
Ishikawa
Krystiana A
KA
Krzysko
Barbara
B
Kowalska-Loth
Aleksandra M
AM
Skrajna
Alicja
A
Czubaty
Agnieszka
A
Girstun
Maja K
MK
Cieplak
Bogdan
B
Lesyng
Krzysztof
K
Staron
D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D053904:DNA Breaks, Single-Stranded; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry*; D010766:Phosphorylation; D016601:RNA-Binding Proteins / Q000737:chemistry*; D000068103:Serine-Arginine Splicing Factors
D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D004247:DNA; D004264:DNA Topoisomerases, Type I