TY - JOUR
T1 - Physical Basis for the Loading of a Bacterial Replicative Helicase onto DNA
AU - Arias-Palomo, Ernesto
AU - Puri, Neha
AU - O'Shea Murray, Valerie L.
AU - Yan, Qianyun
AU - Berger, James M.
N1 - Funding Information:
The authors are grateful to: the EM platforms from Johns Hopkins University, the University of Virginia, Vanderbilt University, the New York Structural Biology center, the University of Leeds, and Israel Sanchez Fernandez (Columbia University) for sample characterization and data analysis; and the EM facilities of the CIB and CNB (CSIC) for support in grid screening and preparation. This work was supported by the NIGMS (to J.M.B. R37-071747), and by the Spanish MINECO/AEI, the ERDF ESF, (to E.A.-P. BFU2017-89143-P and RYC-2015-19059). We acknowledge Diamond for access and support of the Cryo-EM facilities at the UK national electron bio-imaging center (eBIC, proposal EM19454), funded by the Wellcome Trust, MRC, and BBSRC. Access to eBIC was supported by iNEXT, grant number 653706, funded by the Horizon 2020 program of the European Commission. E.A.-P. N.P. and J.M.B. conceived this study. N.P. and E.A.-P. purified the proteins. E.A.-P. performed the cryo-EM experiments, structure determination, and refinement; J.M.B. assisted with model building and data analysis. N.P. carried out the biochemical assays. V.L.O.M. participated in the initial set up of the biochemical experiments. Q.Y. generated some mutants used in this work. E.A.-P. N.P. and J.M.B. wrote the manuscript. The authors declare no competing interests.
Funding Information:
The authors are grateful to: the EM platforms from Johns Hopkins University, the University of Virginia, Vanderbilt University, the New York Structural Biology center, the University of Leeds, and Israel Sanchez Fernandez (Columbia University) for sample characterization and data analysis; and the EM facilities of the CIB and CNB (CSIC) for support in grid screening and preparation. This work was supported by the NIGMS (to J.M.B., R37-071747 ), and by the Spanish MINECO/AEI , the ERDF ESF , (to E.A.-P., BFU2017-89143-P and RYC-2015-19059 ). We acknowledge Diamond for access and support of the Cryo-EM facilities at the UK national electron bio-imaging center (eBIC, proposal EM19454), funded by the Wellcome Trust , MRC , and BBSRC . Access to eBIC was supported by iNEXT , grant number 653706 , funded by the Horizon 2020 program of the European Commission .
Publisher Copyright:
© 2019 Elsevier Inc.
PY - 2019/4/4
Y1 - 2019/4/4
N2 - In cells, dedicated AAA+ ATPases deposit hexameric, ring-shaped helicases onto DNA to initiate chromosomal replication. To better understand the mechanisms by which helicase loading can occur, we used cryo-EM to determine sub-4-Å-resolution structures of the E. coli DnaB⋅DnaC helicase⋅loader complex with nucleotide in pre- and post-DNA engagement states. In the absence of DNA, six DnaC protomers latch onto and crack open a DnaB hexamer using an extended N-terminal domain, stabilizing this conformation through nucleotide-dependent ATPase interactions. Upon binding DNA, DnaC hydrolyzes ATP, allowing DnaB to isomerize into a topologically closed, pre-translocation state competent to bind primase. Our data show how DnaC opens the DnaB ring and represses the helicase prior to DNA binding and how DnaC ATPase activity is reciprocally regulated by DnaB and DNA. Comparative analyses reveal how the helicase loading mechanism of DnaC parallels and diverges from homologous AAA+ systems involved in DNA replication and transposition. Arias-Palomo et al. present the cryo-EM structures of a replicative bacterial helicase-loader complex (E. coli DnaBC) in pre- and post-loading states, revealing how the loader breaks the helicase ring to deposit it at the origin of replication and how ssDNA engagement closes and activates the helicase.
AB - In cells, dedicated AAA+ ATPases deposit hexameric, ring-shaped helicases onto DNA to initiate chromosomal replication. To better understand the mechanisms by which helicase loading can occur, we used cryo-EM to determine sub-4-Å-resolution structures of the E. coli DnaB⋅DnaC helicase⋅loader complex with nucleotide in pre- and post-DNA engagement states. In the absence of DNA, six DnaC protomers latch onto and crack open a DnaB hexamer using an extended N-terminal domain, stabilizing this conformation through nucleotide-dependent ATPase interactions. Upon binding DNA, DnaC hydrolyzes ATP, allowing DnaB to isomerize into a topologically closed, pre-translocation state competent to bind primase. Our data show how DnaC opens the DnaB ring and represses the helicase prior to DNA binding and how DnaC ATPase activity is reciprocally regulated by DnaB and DNA. Comparative analyses reveal how the helicase loading mechanism of DnaC parallels and diverges from homologous AAA+ systems involved in DNA replication and transposition. Arias-Palomo et al. present the cryo-EM structures of a replicative bacterial helicase-loader complex (E. coli DnaBC) in pre- and post-loading states, revealing how the loader breaks the helicase ring to deposit it at the origin of replication and how ssDNA engagement closes and activates the helicase.
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U2 - 10.1016/j.molcel.2019.01.023
DO - 10.1016/j.molcel.2019.01.023
M3 - Article
C2 - 30797687
AN - SCOPUS:85063753774
SN - 1097-2765
VL - 74
SP - 173-184.e4
JO - Molecular cell
JF - Molecular cell
IS - 1
ER -