TY - JOUR
T1 - Strand separation establishes a sustained lock at the Tus-Ter replication fork barrier
AU - Berghuis, Bojk A.
AU - Dulin, David
AU - Xu, Zhi Qiang
AU - Van Laar, Theo
AU - Cross, Bronwen
AU - Janissen, Richard
AU - Jergic, Slobodan
AU - Dixon, Nicholas E.
AU - Depken, Martin
AU - Dekker, Nynke H.
N1 - Accepted Author Manuscript
PY - 2015
Y1 - 2015
N2 - The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand separation on the blocking (nonpermissive) side of Tus-Ter triggers roadblock formation, but biochemical evidence also suggests roles for protein-protein interactions. Here DNA unzipping experiments demonstrate that nonpermissively oriented Tus-Ter forms a tight lock in the absence of replicative proteins, whereas permissively oriented Tus-Ter allows nearly unhindered strand separation. Quantifying the lock strength reveals the existence of several intermediate lock states that are impacted by mutations in the lock domain but not by mutations in the DNA-binding domain. Lock formation is highly specific and exceeds reported in vivo efficiencies. We postulate that protein-protein interactions may actually hinder, rather than promote, proper lock formation.
AB - The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand separation on the blocking (nonpermissive) side of Tus-Ter triggers roadblock formation, but biochemical evidence also suggests roles for protein-protein interactions. Here DNA unzipping experiments demonstrate that nonpermissively oriented Tus-Ter forms a tight lock in the absence of replicative proteins, whereas permissively oriented Tus-Ter allows nearly unhindered strand separation. Quantifying the lock strength reveals the existence of several intermediate lock states that are impacted by mutations in the lock domain but not by mutations in the DNA-binding domain. Lock formation is highly specific and exceeds reported in vivo efficiencies. We postulate that protein-protein interactions may actually hinder, rather than promote, proper lock formation.
KW - DNA-binding proteins
KW - nucleic acids
KW - single-molecule biophysics
UR - http://resolver.tudelft.nl/uuid:376b7dc9-92d7-4ee7-9abf-daa1c33613f4
UR - http://www.scopus.com/inward/record.url?scp=84937729550&partnerID=8YFLogxK
U2 - 10.1038/nchembio.1857
DO - 10.1038/nchembio.1857
M3 - Article
C2 - 26147356
SN - 1552-4450
VL - 11
SP - 579
EP - 585
JO - Nature Chemical Biology
JF - Nature Chemical Biology
ER -