R.KpnI, an HNH superfamily REase, exhibits differential discrimination at non-canonical sequences in the presence of Ca2+ and Mg2+

Nagaraja

2013

Microbiol Mol Biol Rev

521

462

SUMMARY Restriction-modification (R-M) systems are ubiquitous and are often considered primitive immune systems in bacteria. Their diversity and prevalence across the prokaryotic kingdom are an indication of their success as a defense mechanism against invading genomes. However, their cellular defense function does not adequately explain the basis for their immaculate specificity in sequence recognition and nonuniform distribution, ranging from none to too many, in diverse species. The present review deals with new developments which provide insights into the roles of these enzymes in other aspects of cellular function. In this review, emphasis is placed on novel hypotheses and various findings that have not yet been dealt with in a critical review. Emerging studies indicate their role in various cellular processes other than host defense, virulence, and even controlling the rate of evolution of the organism. We also discuss how R-M systems could have successfully evolved and be involved in additional cellular portfolios, thereby increasing the relative fitness of their hosts in the population.

Section: Promiscuity In Cofactor Utilization and Substrate Specificitymentioning

confidence: 99%

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

Nagaraja

2013

Microbiol Mol Biol Rev

521

462

“…The REase exhibits remarkable versatility in substrate recognition and cofactor utilization, whereas the cognate MTase is very site-specific (14). In addition to its recognition sequence, GGTACC, the enzyme binds and cleaves a number of noncanonical sequences (i.e., TGTACC, GTTACC, GATACC, GGAACC, GGTCCC, GGTATC, GGTACG, GGTACT), with the binding affinity ranging from 8 to 35 nM (14,15). Accordingly, many of these sites are cleaved efficiently with k cat values varying from 0.06 to 0.15 min −1 vs. 4.3 min −1 for canonical sites (15).…”

mentioning

confidence: 99%

“…In addition to its recognition sequence, GGTACC, the enzyme binds and cleaves a number of noncanonical sequences (i.e., TGTACC, GTTACC, GATACC, GGAACC, GGTCCC, GGTATC, GGTACG, GGTACT), with the binding affinity ranging from 8 to 35 nM (14,15). Accordingly, many of these sites are cleaved efficiently with k cat values varying from 0.06 to 0.15 min −1 vs. 4.3 min −1 for canonical sites (15). The promiscuous activity of the enzyme is directed by a number of cofactors.…”

mentioning

confidence: 99%

“…The promiscuous activity of the enzyme is directed by a number of cofactors. In the presence of the physiological levels of Mg 2+ , the most abundant cofactor present in the cell (16) and typically required for the activity of REases, R.KpnI discriminates poorly between the canonical and noncanonical sequences, indicating that the promiscuity is an intrinsic property of the enzyme (15). It is intriguing why promiscuity is manifested and what would be its biological significance for the organism.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Promiscuous restriction is a cellular defense strategy that confers fitness advantage to bacteria

Proc. Natl. Acad. Sci. U.S.A.

Nagamalleswari

Nagaraja

2012

Most bacterial genomes harbor restriction–modification systems, encoding a REase and its cognate MTase. On attack by a foreign DNA, the REase recognizes it as nonself and subjects it to restriction. Should REases be highly specific for targeting the invading foreign DNA? It is often considered to be the case. However, when bacteria harboring a promiscuous or high-fidelity variant of the REase were challenged with bacteriophages, fitness was maximal under conditions of catalytic promiscuity. We also delineate possible mechanisms by which the REase recognizes the chromosome as self at the noncanonical sites, thereby preventing lethal dsDNA breaks. This study provides a fundamental understanding of how bacteria exploit an existing defense system to gain fitness advantage during a host–parasite coevolutionary “arms race.”

“…The properties include prolific promiscuous activity in the presence of Mg 2ϩ which is further enhanced with Mn 2ϩ , efficient site specific high fidelity DNA cleavage when Ca 2ϩ is used instead of Mg 2ϩ , and suppression of the promiscuous cleavage activity in presence of Ca 2ϩ (21). Kinetic studies revealed that the Ca 2ϩ -mediated exquisite specificity is achieved at the step of DNA cleavage (22).…”

mentioning

confidence: 99%

Dual Role for Zn2+ in Maintaining Structural Integrity and Inducing DNA Sequence Specificity in a Promiscuous Endonuclease

Matheshwaran

Journal of Biological Chemistry

Ghosh

et al. 2007

induce promiscuous cleavage by the enzyme, whereas Ca 2؉ -bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg 2؉ -and Mn 2؉ -mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase.