HNH proteins are a widespread component of phage DNA packaging machines

Kala, Smriti; Cumby, Nichole; Sadowski, Paul D.; Hyder, Batool Z.; Kanelis, Voula; Davidson, Alan R.; Maxwell, Karen L.

doi:10.1073/pnas.1320952111

Cited by 120 publications

(117 citation statements)

References 35 publications

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“…HNH motifcontaining endonuclease proteins may interact with phage terminase proteins to promote phage DNA packaging and maturation (31). For example, a recent report showed that gp74 (an HNH endonuclease) of phage HK97 interacts with its terminase protein (32) and that the HNH motif of endonuclease is essential for interacting with terminase and completion of DNA packaging. Also, lambda gpFI, an endonuclease, interacts with lambda terminase (16).…”

Section: Discussionmentioning

confidence: 99%

The Protein Interactome of Mycobacteriophage Giles Predicts Functions for Unknown Proteins

et al. 2015

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Mycobacteriophages are viruses that infect mycobacterial hosts and are prevalent in the environment. Nearly 700 mycobacteriophage genomes have been completely sequenced, revealing considerable diversity and genetic novelty. Here, we have determined the protein complement of mycobacteriophage Giles by mass spectrometry and mapped its genome-wide protein interactome to help elucidate the roles of its 77 predicted proteins, 50% of which have no known function. About 22,000 individual yeast twohybrid (Y2H) tests with four different Y2H vectors, followed by filtering and retest screens, resulted in 324 reproducible proteinprotein interactions, including 171 (136 nonredundant) high-confidence interactions. The complete set of high-confidence interactions among Giles proteins reveals new mechanistic details and predicts functions for unknown proteins. The Giles interactome is the first for any mycobacteriophage and one of just five known phage interactomes so far. Our results will help in understanding mycobacteriophage biology and aid in development of new genetic and therapeutic tools to understand Mycobacterium tuberculosis. IMPORTANCEMycobacterium tuberculosis causes over 9 million new cases of tuberculosis each year. Mycobacteriophages, viruses of mycobacterial hosts, hold considerable potential to understand phage diversity, evolution, and mycobacterial biology, aiding in the development of therapeutic tools to control mycobacterial infections. The mycobacteriophage Giles protein-protein interaction network allows us to predict functions for unknown proteins and shed light on major biological processes in phage biology. For example, Giles gp76, a protein of unknown function, is found to associate with phage packaging and maturation. The functions of mycobacteriophage-derived proteins may suggest novel therapeutic approaches for tuberculosis. Our ORFeome clone set of Giles proteins and the interactome data will be useful resources for phage interactomics.T he continuous emergence of bacterial pathogens resistant to antibiotics is an increasing medical problem (1). Mycobacterium tuberculosis is prominent among these pathogens, with over 9 million new cases of tuberculosis reported each year. There is an urgent need for alternate ways to control M. tuberculosis infections, and one potential strategy involves using mycobacteriophages for prophylaxis or therapy (2). The emergence of extensively drug-resistant (XDR) and totally drug-resistant (TDR) strains of M. tuberculosis, both of which are especially difficult to control (3), has spurred renewed interest in the therapeutic use of bacteriophages.Mycobacteriophages are known to infect many different species of both fast-and slow-growing mycobacteria, including M. tuberculosis and Mycobacterium smegmatis (4, 5). Over the past decade, thousands of mycobacteriophages have been isolated, and hundreds have been completely sequenced (http://phagesdb.org/). Mycobacteriophage genomes are highly mosaic due to horizontal genetic exchange (6-8) but can be grouped into 2...

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Section: Discussionmentioning

confidence: 99%

The Protein Interactome of Mycobacteriophage Giles Predicts Functions for Unknown Proteins

et al. 2015

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“…Another HNHE, gp74 was discovered from E. coli phage HK97 that cleaves λ DNA into small fragments in Ni 2+ or Zn 2+ buffer (26,27). Gp74 was shown to be associated with HK97 terminase large and small subunits (TerL/S) complex and stimulated cleavage/nicking at the cohesive end site (cos) (28). It was speculated that TerL-S enzyme may cleave one strand and gp74 may nick the opposite strand.…”

Section: Naturally Occurring Hnhe Associated With Bacterial Mobile Gementioning

confidence: 99%

“…It was convincingly demonstrated that gp74 was involved in DNA packaging and morphogenesis since HK97 gp74 amber mutant (74am) produced only empty prohead, but generated normal tails structures that can complement tail-deficient mutants. Furthermore, gp74 amber phage can be rescued by in vivo complementation when gp74 is provided in trans from a plasmid (28). To analyze the cleavage/nicking sites of gp74, we expressed and purified gp74 (see Supplementary Material).…”

Section: Naturally Occurring Hnhe Associated With Bacterial Mobile Gementioning

confidence: 99%

Sequence-specific DNA nicking endonucleases

2015

Biomolecular Concepts

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A group of small HNH nicking endonucleases (NEases) was discovered recently from phage or prophage genomes that nick double-stranded DNA sites ranging from 3 to 5 bp in the presence of Mg2+ or Mn2+. The cosN site of phage HK97 contains a gp74 nicking site AC↑CGC, which is similar to AC↑CGR (R=A/G) of N.ϕGamma encoded by Bacillus phage Gamma. A minimal nicking domain of 76 amino acid residues from N.ϕGamma could be fused to other DNA binding partners to generate chimeric NEases with new specificities. The biological roles of a few small HNH endonucleases (HNHE, gp74 of HK97, gp37 of ϕSLT, ϕ12 HNHE) have been demonstrated in phage and pathogenicity island DNA packaging. Another group of NEases with 3- to 7-bp specificities are either natural components of restriction systems or engineered from type IIS restriction endonucleases. A phage group I intron-encoded HNH homing endonucleases, I-PfoP3I was found to nick DNA sites of 14-16 bp. I-TslI encoded by T7-like ΦI appeared to nick DNA sites with a 9-bp core sequence. DNA nicking and labeling have been applied to optical mapping to aid genome sequence assembly and detection of large insertion/deletion mutations in genomic DNA of cancer cells. Nicking enzyme-mediated amplification reaction has been applied to rapid diagnostic testing of influenza A and B in clinical setting and for construction of DNA-based Boolean logic gates. The clustered regularly interspaced short palindromic repeats-ribonucleoprotein complex consisting of engineered Cas9 nickases in conjunction with tracerRNA:crRNA or a single-guide RNA have been successfully used in genome modifications.

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“…This mechanism was confirmed recently for Microplitis demolitor BV (MdBV) by inactivation of two nudiviruslike tyrosine recombinase genes (Vlf1 and Int-1) using RNA interference, which indeed resulted in impairment of circle formation [18 ]. Circularization probably occurs in combination with entry of replicated DNA into the nucleocapsids [7], as found for many viruses for which concatemers of genomes produced during DNA replication are separated during encapsidation (for example phages [34] and herpesviruses [35]). …”

Section: Chromosomal Amplification (Drosophila Chorion Genes «Onion Smentioning

confidence: 56%