Assembly of a polymeric chain of SUMO1 on human topoisomerase I in vitro

Meiluen Yang, Chia Tse Hsu, Chun Yuan Ting, Leroy-Fong Liu, Jaulang Hwang

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

Human (h) DNA topoisomerase I has been identified as a major SUMO1 target in camptothecin-treated cells. In response to TOP1-mediated DNA damage induced by camptothecin, multiple SUMO1 molecules are conjugated to the N-terminal domain of a single TOP1 molecule. To investigate the molecular mechanism of SUMO1 conjugation to TOP1, an in vitro system using purified SAE1/2, Ubc9, SUMO1, and TOP1 peptides was developed. Consistent with results from in vivo studies, multiple SUMO1 molecules were found to be conjugated to the N-terminal domain of a single TOP1 molecule. Systematic analysis has identified a single major SUMO1 conjugation site located between amino acid residues 110 and 125 that contains a single lysine residue at 117 (Lys-117). Using a short peptide spanning this region, we showed that a poly-SUMO1 chain was assembled in this peptide at Lys-117. Interestingly, a Ubc9-poly-SUMO1 intermediate had accumulated to a high level when the sumoylation assay was performed in the absence of hTOP1 substrate, suggesting a possibility that the poly-SUMO1 chain is formed on Ubc9 first and then transferred en bloc onto hTOP1. This is the first definitive demonstration of the assembly of a poly-SUMO1 chain on protein substrate. These results offer new insight into hTOP1 polysumoylation in response to TOP1-mediated DNA damage and may have general implications in protein polysumoylation.

Original languageEnglish
Pages (from-to)8264-8274
Number of pages11
JournalJournal of Biological Chemistry
Volume281
Issue number12
DOIs
Publication statusPublished - Mar 24 2006
Externally publishedYes

Fingerprint

Type I DNA Topoisomerase
Camptothecin
Peptides
Lysine
DNA Damage
Molecules
Sumoylation
DNA
Proteins
Substrates
Amino Acids
Assays
Demonstrations
Cells
In Vitro Techniques

ASJC Scopus subject areas

  • Biochemistry

Cite this

Assembly of a polymeric chain of SUMO1 on human topoisomerase I in vitro. / Yang, Meiluen; Hsu, Chia Tse; Ting, Chun Yuan; Liu, Leroy-Fong; Hwang, Jaulang.

In: Journal of Biological Chemistry, Vol. 281, No. 12, 24.03.2006, p. 8264-8274.

Research output: Contribution to journalArticle

Yang, Meiluen ; Hsu, Chia Tse ; Ting, Chun Yuan ; Liu, Leroy-Fong ; Hwang, Jaulang. / Assembly of a polymeric chain of SUMO1 on human topoisomerase I in vitro. In: Journal of Biological Chemistry. 2006 ; Vol. 281, No. 12. pp. 8264-8274.
@article{4d1285bb894e4a158f846668bc75bc82,
title = "Assembly of a polymeric chain of SUMO1 on human topoisomerase I in vitro",
abstract = "Human (h) DNA topoisomerase I has been identified as a major SUMO1 target in camptothecin-treated cells. In response to TOP1-mediated DNA damage induced by camptothecin, multiple SUMO1 molecules are conjugated to the N-terminal domain of a single TOP1 molecule. To investigate the molecular mechanism of SUMO1 conjugation to TOP1, an in vitro system using purified SAE1/2, Ubc9, SUMO1, and TOP1 peptides was developed. Consistent with results from in vivo studies, multiple SUMO1 molecules were found to be conjugated to the N-terminal domain of a single TOP1 molecule. Systematic analysis has identified a single major SUMO1 conjugation site located between amino acid residues 110 and 125 that contains a single lysine residue at 117 (Lys-117). Using a short peptide spanning this region, we showed that a poly-SUMO1 chain was assembled in this peptide at Lys-117. Interestingly, a Ubc9-poly-SUMO1 intermediate had accumulated to a high level when the sumoylation assay was performed in the absence of hTOP1 substrate, suggesting a possibility that the poly-SUMO1 chain is formed on Ubc9 first and then transferred en bloc onto hTOP1. This is the first definitive demonstration of the assembly of a poly-SUMO1 chain on protein substrate. These results offer new insight into hTOP1 polysumoylation in response to TOP1-mediated DNA damage and may have general implications in protein polysumoylation.",
author = "Meiluen Yang and Hsu, {Chia Tse} and Ting, {Chun Yuan} and Leroy-Fong Liu and Jaulang Hwang",
year = "2006",
month = "3",
day = "24",
doi = "10.1074/jbc.M510364200",
language = "English",
volume = "281",
pages = "8264--8274",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "12",

}

TY - JOUR

T1 - Assembly of a polymeric chain of SUMO1 on human topoisomerase I in vitro

AU - Yang, Meiluen

AU - Hsu, Chia Tse

AU - Ting, Chun Yuan

AU - Liu, Leroy-Fong

AU - Hwang, Jaulang

PY - 2006/3/24

Y1 - 2006/3/24

N2 - Human (h) DNA topoisomerase I has been identified as a major SUMO1 target in camptothecin-treated cells. In response to TOP1-mediated DNA damage induced by camptothecin, multiple SUMO1 molecules are conjugated to the N-terminal domain of a single TOP1 molecule. To investigate the molecular mechanism of SUMO1 conjugation to TOP1, an in vitro system using purified SAE1/2, Ubc9, SUMO1, and TOP1 peptides was developed. Consistent with results from in vivo studies, multiple SUMO1 molecules were found to be conjugated to the N-terminal domain of a single TOP1 molecule. Systematic analysis has identified a single major SUMO1 conjugation site located between amino acid residues 110 and 125 that contains a single lysine residue at 117 (Lys-117). Using a short peptide spanning this region, we showed that a poly-SUMO1 chain was assembled in this peptide at Lys-117. Interestingly, a Ubc9-poly-SUMO1 intermediate had accumulated to a high level when the sumoylation assay was performed in the absence of hTOP1 substrate, suggesting a possibility that the poly-SUMO1 chain is formed on Ubc9 first and then transferred en bloc onto hTOP1. This is the first definitive demonstration of the assembly of a poly-SUMO1 chain on protein substrate. These results offer new insight into hTOP1 polysumoylation in response to TOP1-mediated DNA damage and may have general implications in protein polysumoylation.

AB - Human (h) DNA topoisomerase I has been identified as a major SUMO1 target in camptothecin-treated cells. In response to TOP1-mediated DNA damage induced by camptothecin, multiple SUMO1 molecules are conjugated to the N-terminal domain of a single TOP1 molecule. To investigate the molecular mechanism of SUMO1 conjugation to TOP1, an in vitro system using purified SAE1/2, Ubc9, SUMO1, and TOP1 peptides was developed. Consistent with results from in vivo studies, multiple SUMO1 molecules were found to be conjugated to the N-terminal domain of a single TOP1 molecule. Systematic analysis has identified a single major SUMO1 conjugation site located between amino acid residues 110 and 125 that contains a single lysine residue at 117 (Lys-117). Using a short peptide spanning this region, we showed that a poly-SUMO1 chain was assembled in this peptide at Lys-117. Interestingly, a Ubc9-poly-SUMO1 intermediate had accumulated to a high level when the sumoylation assay was performed in the absence of hTOP1 substrate, suggesting a possibility that the poly-SUMO1 chain is formed on Ubc9 first and then transferred en bloc onto hTOP1. This is the first definitive demonstration of the assembly of a poly-SUMO1 chain on protein substrate. These results offer new insight into hTOP1 polysumoylation in response to TOP1-mediated DNA damage and may have general implications in protein polysumoylation.

UR - http://www.scopus.com/inward/record.url?scp=33646353695&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33646353695&partnerID=8YFLogxK

U2 - 10.1074/jbc.M510364200

DO - 10.1074/jbc.M510364200

M3 - Article

VL - 281

SP - 8264

EP - 8274

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 12

ER -