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001-es BibID:	BIBFORM031395
035-os BibID:	(Scopus)70349229163 (WoS)000271341400011 (PMID)19682999
Első szerző:	Pingoud, Vera
Cím:	On the divalent metal ion dependence of DNA cleavage by restriction endonucleases of the EcoRI family / Vera Pingoud, Wolfgang Wende, Peter Friedhoff, Monika Reuter, Jürgen Alves, Albert Jeltsch, Letif Mones, Monika Fuxreiter, Alfred Pingoud
Dátum:	2009
Megjegyzések:	Restriction endonucleases of the PD...D/EXK family need Mg(2+) for DNA cleavage. Whereas Mg(2+) (or Mn(2+)) promotes catalysis, Ca(2+) (without Mg(2+)) only supports DNA binding. The role of Mg(2+) in DNA cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg(2+) involved in catalysis. To address this problem, we measured the Mg(2+) and Mn(2+) concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me(2+) per active site. DNA cleavage experiments were carried out at various Mg(2+) and Mn(2+) concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg(2+) and Mn(2+) concentration dependence. In general, the Mg(2+) concentration optimum (between approximately 1 and 10 mM) is higher than the Mn(2+) concentration optimum (between approximately 0.1 and 1 mM). At still higher Mg(2+) or Mn(2+) concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca(2+). Based on these results, we propose that one Mg(2+) or Mn(2+) is critical for restriction enzyme activation, and binding of a second Me(2+) plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg(2+) or Mn(2+) mainly leads to an increase in K(m), such that the inhibitory effect of excess Mg(2+) or Mn(2+) can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me(2+) binding to these enzymes.
Tárgyszavak:	Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban külföldön készült közlemény
Megjelenés:	Journal of molecular biology. - 393 : 1 (2009), p. 140-160. -
További szerzők:	Wende, Wolfgang Friedhoff, Peter Reuter Mónika Alves, Jürgen Jeltsch, Albert Mones, Letif Fuxreiter Mónika (1969-) (kutató vegyész) Pingoud, Alfred
Internet cím:	Intézményi repozitóriumban (DEA) tárolt változat DOI
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