Multiple roles for divalent metal ions in DNA transposition: distinct stages of Tn10 transposition have different Mg2+ requirements.

Abstract

Tn10 transposition takes place by a non‐replicative mechanism in which the transposon is excised from donor DNA and integrated into a target site. Mg2+ is an essential cofactor in this reaction. We have examined the Mg2+ requirements at various steps in Tn10 transposition. Results presented here demonstrate that Tn10 excision can occur efficiently at a 16‐fold lower Mg2+ concentration than strand transfer and that, at Mg2+ concentrations in the range of 60‐fold below the wildt‐ype optimum, double strand cleavage events at the two transposon ends are completely uncoupled. These experiments identify specific breakpoints in Tn10 transposition which are sensitive to Mg2+ concentration. Whereas the uncoupling of double strand cleavage events at the two transposon ends most likely reflects the inability of two separate IS10 transposase monomers in the synaptic complex to bind Mg2+, the uncoupling of transposon excision from strand transfer is expected to reflect either a conformational change in the active site or the existence of an Mg2+ binding site which functions specifically in target interactions. We also show that Mn2+ relaxes target specificity in Tn10 transposition and suppresses a class of mutants which are blocked specifically for integration. These observations can be explained by a model in which sequence‐specific target site binding is tightly coupled to a conformational change in the synaptic complex which is required for catalysis of strand transfer.

Divalent Metal Ion Differentially Regulates the Sequential Nicking Reactions of the GIY-YIG Homing Endonuclease I-BmoI

“…Similar roles for metal ions in promoting protein-DNA interactions and conformational changes required for efficient catalysis have been dissected in other enzyme systems where a single active site is used sequentially to perform multiple reactions [57]. Notably, sub-optimal magnesium concentrations can uncouple excision and strand transfer events, and manganese can reduce target site specificity of the Tn10 transposase [57].…”

“…Notably, sub-optimal magnesium concentrations can uncouple excision and strand transfer events, and manganese can reduce target site specificity of the Tn10 transposase [57]. However, the modular structure of I-BmoI and other GIY-YIG homing endonucleases resembles that of Type IIs restriction enzymes, including the well-characterized FokI that is a monomer in solution but transiently dimerizes to generate a DSB [58], [59].…”

Metal-Dependent Conformational Activation Explains Highly Promutagenic Replication across O6-Methylguanine by Human DNA Polymerase β

“…52,53,59 DNA polymerases probably utilize the catalytic Mg 2+ , which is highly sensitive to the presence of active-site mutations, base pair mismatch, and suboptimal substrates, 69 to increase substrate specificity and replication fidelity. The replacement of Mg 2+ with Mn 2+ , which is more tolerant of active-site distortions and abnormal substrates than Mg 2+ , 69 could stimulate the binding and the subsequent coordination of the catalytic metal ion during nucleotide misincorporation, thereby faciliating the incorporation of otherwise unfavorable substrates and decreasing replication fidelity of DNA polymerase.…”

Strand-specific Contacts and Divalent Metal Ion Regulate Double-strand Break Formation by the GIY-YIG Homing Endonuclease I-BmoI

“…Similar "uncoupling" of cleavage steps by limiting metal ion availability has been reported in a number of transposition systems. 44,45 In the case of I-BmoI, it is possible that the metal ion required for top-strand nicking promotes or stabilizes a rearrangement in the I-BmoI catalytic domain, or DNA substrate in the I-BmoI-thyA complex, to reposition the active site at the top strand scissile phosphate. Indeed, our observation that top-strand nicking specificity is completely switched on substrates that lack the critical G-C base-pair at position −2 is consistent with the notion that divalent metal ion may mediate a contact between the G or C base at position −2 and a residue(s) in the catalytic domain, aiding to position the catalytic domain on the top strand.…”