Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase
[EN] During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (~50 pN). ; We thank Stephan Grill laboratory (MPI-CBG, Dresden) for help with data collection and E. Galburt, M. Manosas and M. De Vega for critical reading of the manuscript. Spanish Ministry of Economy and Competitiveness [BFU2011-29038 to J.L.C., BFU2013-44202 to J.M.V., BFU2011-23645 to M.S., FIS2010-17440, GR35/10-A920GR35/10-A-911 to F.J.C., MAT2013-49455-EXP to J.R.A.-G. and BFU2012-31825 to B.I.]; Regional Government of Madrid [S2009/MAT 1507 to J.L.C. and CDS2007-0015 to M.S.]; European Molecular Biology Organization [ASTF 276-2012 to J.M.L.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2012-31825 to B.I.]. ; Morin, J.; Cao, F.; Lázaro, J.; Arias-Gonzalez, JR.; Valpuesta, J.; Carrascosa, J.; Salas, M. (2015). Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase. Nucleic Acids Research. 43(7):3643-3652. https://doi.org/10.1093/nar/gkv204 ; S ; 3643 ; 3652 ; 43 ...