Naturally-occurring, dually-functional fusions between restriction endonucleases and regulatory proteinsReport as inadecuate




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BMC Evolutionary Biology

, 13:218

Genome evolution and evolutionary systems biology

Abstract

BackgroundRestriction-modification RM systems appear to play key roles in modulating gene flow among bacteria and archaea. Because the restriction endonuclease REase is potentially lethal to unmethylated new host cells, regulation to ensure pre-expression of the protective DNA methyltransferase MTase is essential to the spread of RM genes. This is particularly true for Type IIP RM systems, in which the REase and MTase are separate, independently-active proteins. A substantial subset of Type IIP RM systems are controlled by an activator-repressor called C protein. In these systems, C controls the promoter for its own gene, and for the downstream REase gene that lacks its own promoter. Thus MTase is expressed immediately after the RM genes enter a new cell, while expression of REase is delayed until sufficient C protein accumulates. To study the variation in and evolution of this regulatory mechanism, we searched for RM systems closely related to the well-studied C protein-dependent PvuII RM system. Unexpectedly, among those found were several in which the C protein and REase genes were fused.

ResultsThe gene for CR.NsoJS138I fusion protein nsoJS138ICR, from the bacterium Niabella soli was cloned, and the fusion protein produced and partially purified. Western blots provided no evidence that, under the conditions tested, anything other than full-length fusion protein is produced. This protein had REase activity in vitro and, as expected from the sequence similarity, its specificity was indistinguishable from that for PvuII REase, though the optimal reaction conditions were different. Furthermore, the fusion was active as a C protein, as revealed by in vivo activation of a lacZ reporter fusion to the promoter region for the nsoJS138ICR gene.

ConclusionsFusions between C proteins and REases have not previously been characterized, though other fusions have such as between REases and MTases. These results reinforce the evidence for impressive modularity among RM system proteins, and raise important questions about the implications of the C-REase fusions on expression kinetics of these RM systems.

KeywordsRestriction-modification systems Restriction endonuclease Gene regulation Fused genes C protein Regulatory evolution AbbreviationsMTaseModification DNA methyltransferase

REaseRestriction endonuclease

RMRestriction-modification

LacZβ-galactosidase

IPTGIsopropythio-β-D-galactoside

ONPGO-nitrophenyl-β-D-thiogalactoside

HRPHorseradish peroxidase

PMSFPhenyl-methyl-sulfonylfluoride

ODOptical density

SDSSodium dodecylsulfate.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2148-13-218 contains supplementary material, which is available to authorized users.

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Author: Jixiao Liang - Robert M Blumenthal

Source: https://link.springer.com/







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