Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy

Adam F. Hastings; Gottfried Otting; Rutger H.A. Folmer; Iain G. Duggin; R. Gerry Wake; Matthew C.J. Wilce; Jacqueline A. Wilce

doi:10.1016/j.bbrc.2005.07.082

Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy

Adam F. Hastings, Gottfried Otting, Rutger H.A. Folmer, Iain G. Duggin, R. Gerry Wake, Matthew C.J. Wilce, Jacqueline A. Wilce^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

3 Citations (Scopus)

Abstract

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in ¹H-¹⁵N correlation spectra of a ¹⁵N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the ¹H-¹⁵N correlations was achieved using a suite of triple resonance NMR experiments with ¹⁵N,¹³C,70% ²H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to ¹H-¹⁵N spectra revealed that the N-termini, α3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.

Original language	English
Pages (from-to)	361-366
Number of pages	6
Journal	Biochemical and Biophysical Research Communications
Volume	335
Issue number	2
DOIs	https://doi.org/10.1016/j.bbrc.2005.07.082
Publication status	Published - 23 Sept 2005

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10.1016/j.bbrc.2005.07.082

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@article{24a20c701adf4a328ccbbc845bcd6e0c,

title = "Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy",

abstract = "Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in 1H-15N correlation spectra of a 15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the 1H-15N correlations was achieved using a suite of triple resonance NMR experiments with 15N,13C,70% 2H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to 1H-15N spectra revealed that the N-termini, α3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.",

keywords = "Duplex DNA, HSQC titration, NMR spectroscopy, Protein-DNA complex, RTP, Structural perturbation, TROSY, Terminator DNA, Winged-helix protein",

author = "Hastings, {Adam F.} and Gottfried Otting and Folmer, {Rutger H.A.} and Duggin, {Iain G.} and Wake, {R. Gerry} and Wilce, {Matthew C.J.} and Wilce, {Jacqueline A.}",

year = "2005",

month = sep,

day = "23",

doi = "10.1016/j.bbrc.2005.07.082",

language = "English",

volume = "335",

pages = "361--366",

journal = "Biochemical and Biophysical Research Communications",

issn = "0006-291X",

publisher = "Elsevier B.V.",

number = "2",

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T1 - Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy

AU - Hastings, Adam F.

AU - Otting, Gottfried

AU - Folmer, Rutger H.A.

AU - Duggin, Iain G.

AU - Wake, R. Gerry

AU - Wilce, Matthew C.J.

AU - Wilce, Jacqueline A.

PY - 2005/9/23

Y1 - 2005/9/23

N2 - Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in 1H-15N correlation spectra of a 15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the 1H-15N correlations was achieved using a suite of triple resonance NMR experiments with 15N,13C,70% 2H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to 1H-15N spectra revealed that the N-termini, α3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.

AB - Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in 1H-15N correlation spectra of a 15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the 1H-15N correlations was achieved using a suite of triple resonance NMR experiments with 15N,13C,70% 2H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to 1H-15N spectra revealed that the N-termini, α3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.

KW - Duplex DNA

KW - HSQC titration

KW - NMR spectroscopy

KW - Protein-DNA complex

KW - RTP

KW - Structural perturbation

KW - TROSY

KW - Terminator DNA

KW - Winged-helix protein

UR - http://www.scopus.com/inward/record.url?scp=23744473897&partnerID=8YFLogxK

U2 - 10.1016/j.bbrc.2005.07.082

DO - 10.1016/j.bbrc.2005.07.082

M3 - Article

SN - 0006-291X

VL - 335

SP - 361

EP - 366

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

IS - 2

ER -

Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy

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