Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element.

Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair. In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins. Here, we describe a unique molecular solution to assemble a functional product from the RNR large subunit gene, nrdA that has been fragmented into two smaller genes by the insertion of mobE, a mobile endonuclease. We show that unique sequences that originated during the mobE insertion and that are present as C- and N-terminal tails on the split NrdA-a and NrdA-b polypeptides, are absolutely essential for enzymatic activity. Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)(2) large subunit necessary for a functional RNR holoenzyme. The tails represent a solution distinct from RNA and protein splicing or programmed DNA rearrangements to restore function from a fragmented coding region and may represent a general mechanism to neutralize fragmentation of essential genes by mobile genetic elements.

Stress-induced inactivation of the Staphylococcus aureus purine biosynthesis repressor leads to hypervirulence.

Staphylococcus aureus is a significant cause of human infection. Here, we demonstrate that mutations in the transcriptional repressor of purine biosynthesis, purR, enhance the pathogenic potential of S. aureus. Indeed, systemic infection with purR mutants causes accelerated mortality in mice, which is due to aberrant up-regulation of fibronectin binding proteins (FnBPs). Remarkably, purR mutations can arise upon exposure of S. aureus to stress, such as an intact immune system. In humans, naturally occurring anti-FnBP antibodies exist that, while not protective against recurrent S. aureus infection, ostensibly protect against hypervirulent S. aureus infections. Vaccination studies support this notion, where anti-Fnb antibodies in mice protect against purR hypervirulence. These findings provide a novel link between purine metabolism and virulence in S. aureus.

Insertion of a homing endonuclease creates a genes-in-pieces ribonucleotide reductase that retains function.

In bacterial and phage genomes, coding regions are sometimes interrupted by self-splicing introns or inteins, which can encode mobility-promoting homing endonucleases. Homing endonuclease genes are also found free-standing (not intron- or intein-encoded) in phage genomes where they are inserted in intergenic regions. One example is the HNH family endonuclease, mobE, inserted between the large (nrdA) and small (nrdB) subunit genes of aerobic ribonucleotide reductase (RNR) of T-even phages T4, RB2, RB3, RB15, and LZ7. Here, we describe an insertion of mobE into the nrdA gene of Aeromonas hydrophila phage Aeh1. The insertion creates a unique genes-in-pieces arrangement, where nrdA is split into two independent genes, nrdA-a and nrdA-b, each encoding cysteine residues that correspond to the active-site residues of uninterrupted NrdA proteins. Remarkably, the mobE insertion does not inactivate NrdA function, although the insertion is not a self-splicing intron or intein. We copurified the NrdA-a, NrdA-b, and NrdB proteins as complex from Aeh1-infected cells and also showed that a reconstituted complex has RNR activity. Class I RNR activity in phage Aeh1 is thus assembled from separate proteins that interact to form a composite active site, demonstrating that the mobE insertion is phenotypically neutral in that its presence as an intervening sequence does not disrupt the function of the surrounding gene.

Strand-specific contacts and divalent metal ion regulate double-strand break formation by the GIY-YIG homing endonuclease I-BmoI.

GIY-YIG homing endonucleases are modular enzymes consisting of a well-defined N-terminal catalytic domain connected to a variable C-terminal DNA-binding domain. Previous studies have revealed that the role of the DNA-binding domain is to recognize and bind intronless DNA substrate, positioning the N-terminal catalytic domain such that it is poised to generate a staggered double-strand break by an unknown mechanism. Interactions of the N-terminal catalytic domain with intronless substrate are therefore a critical step in the reaction pathway but have been difficult to define. Here, we have taken advantage of the reduced activity of I-BmoI, an isoschizomer of the well-studied bacteriophage T4 homing endonuclease I-TevI, to examine double-strand break formation by I-BmoI. We present evidence demonstrating that I-BmoI generates a double-strand break by two sequential but chemically independent nicking reactions where divalent metal ion is a limiting factor in top-strand nicking. We also show by in-gel footprinting that contacts by the I-BmoI catalytic domain induce significant minor groove DNA distortions that occur independently of bottom-strand nicking. Bottom-strand contacts are critical for accurate top-strand nicking, whereas top-strand contacts have little influence on the accuracy of bottom-strand nicking. We discuss our results in the context of current models of GIY-YIG endonuclease function, with emphasis on the role of divalent metal ion and strand-specific contacts in regulating the activity of a single active site to generate a staggered double-strand break.