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Articles by S. Shuman
Total Records ( 4 ) for S. Shuman
  K. M Sinha , M. C Unciuleac , M. S Glickman and S. Shuman
 

The resection of DNA double-strand breaks (DSBs) in bacteria is a motor-driven process performed by a multisubunit helicase–nuclease complex: either an Escherichia coli-type RecBCD enzyme or a Bacillus-type AddAB enzyme. Here we identify mycobacterial AdnAB as the founder of a new family of heterodimeric helicase–nucleases with distinctive properties. The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal nuclease module. The AdnAB ATPase is triggered by dsDNA with free ends and the energy of ATP hydrolysis is coupled to DSB end resection by the AdnAB nuclease. The mycobacterial nonhomologous end-joining (NHEJ) protein Ku protects DSBs from resection by AdnAB. We find that AdnAB incises ssDNA by measuring the distance from the free 5' end to dictate the sites of cleavage, which are predominantly 5 or 6 nucleotides (nt) from the 5' end. The "molecular ruler" of AdnAB is regulated by ATP, which elicits an increase in ssDNA cleavage rate and a distal displacement of the cleavage sites 16–17 nt from the 5' terminus. AdnAB is a dual nuclease with a clear division of labor between the subunits. Mutations in the nuclease active site of the AdnB subunit ablate the ATP-inducible cleavages; the corresponding changes in AdnA abolish ATP-independent cleavage. Complete suppression of DSB end resection requires simultaneous mutation of both subunit nucleases. The nuclease-null AdnAB is a helicase that unwinds linear plasmid DNA without degrading the displaced single strands. Mutations of the phosphohydrolase active site of the AdnB subunit ablate DNA-dependent ATPase activity, DSB end resection, and ATP-inducible ssDNA cleavage; the equivalent mutations of the AdnA subunit have comparatively little effect. AdnAB is a novel signature of the Actinomycetales taxon. Mycobacteria are exceptional in that they encode both AdnAB and RecBCD, suggesting the existence of alternative end-resecting motor–nuclease complexes.

  N Keppetipola , R Jain , B Meineke , M Diver and S. Shuman
 

tRNA anticodon damage inflicted by secreted ribotoxins such as Kluyveromyces lactis -toxin and bacterial colicins underlies a rudimentary innate immune system that distinguishes self from nonself species. The intracellular expression of -toxin (a 232-amino acid polypeptide) arrests the growth of Saccharomyces cerevisiae by incising a single RNA phosphodiester 3' of the modified wobble base of tRNAGlu. Fungal -toxin bears no primary structure similarity to any known nuclease and has no plausible homologs in the protein database. To gain insight to -toxin's mechanism, we tested the effects of alanine mutations at 62 basic, acidic, and polar amino acids on ribotoxin activity in vivo. We thereby identified 22 essential residues, including 10 lysines, seven arginines, three glutamates, one cysteine, and one histidine (His209, the only histidine present in -toxin). Structure–activity relations were gleaned from the effects of 44 conservative substitutions. Recombinant tag-free -toxin, a monomeric protein, incised an oligonucleotide corresponding to the anticodon stem–loop of tRNAGlu at a single phosphodiester 3' of the wobble uridine. The anticodon nuclease was metal independent. RNA cleavage was abolished by ribose 2'-H and 2'-F modifications of the wobble uridine. Mutating His209 to alanine, glutamine, or asparagine abolished nuclease activity. We propose that -toxin catalyzes an RNase A-like transesterification reaction that relies on His209 and a second nonhistidine side chain as general acid–base catalysts.

  B Schwer , S Schneider , Y Pei , A Aronova and S. Shuman
 

The Spt5-Spt4 complex regulates early transcription elongation by RNA polymerase II and has an imputed role in pre-mRNA processing via its physical association with mRNA capping enzymes. Here we characterize the Schizosaccharomyces pombe core Spt5-Spt4 complex as a heterodimer and map a trypsin-resistant Spt4-binding domain within the Spt5 subunit. A genetic analysis of Spt4 in S. pombe revealed it to be inessential for growth at 25°C–30°C but critical at 37°C. These results echo the conditional spt4 growth phenotype in budding yeast, where we find that Saccharomyces cerevisiae and S. pombe Spt4 are functionally interchangeable. Complementation of S. cerevisiae spt4 and a two-hybrid assay for Spt4-Spt5 interaction provided a readout of the effects of 33 missense and truncation mutations on S. pombe Spt4 function in vivo, which were interpreted in light of the recent crystal structure of S. cerevisiae Spt4 fused to a fragment of Spt5. Our results highlight the importance of the Spt4 Zn2+-binding residues—Cys12, Cys15, Cys29, and Asp32—and of Ser57, a conserved constituent of the Spt4-Spt5 interface. The 990-amino acid S. pombe Spt5 protein has an exceptionally regular carboxyl-terminal domain (CTD) composed of 18 nonapeptide repeats. We find that as few as three nonamer repeats sufficed for S. pombe growth, but only when Spt4 was present. Synthetic lethality of the spt51-835 spt4 double mutant at 34°C suggests that interaction of Spt4 with the central domain of Spt5 overlaps functionally with the Spt5 CTD.

  S. Shuman
 

DNA ligases seal 5'-PO4 and 3'-OH polynucleotide ends via three nucleotidyl transfer steps involving ligase-adenylate and DNA-adenylate intermediates. DNA ligases are essential guardians of genomic integrity, and ligase dysfunction underlies human genetic disease syndromes. Crystal structures of DNA ligases bound to nucleotide and nucleic acid substrates have illuminated how ligase reaction chemistry is catalyzed, how ligases recognize damaged DNA ends, and how protein domain movements and active-site remodeling are used to choreograph the end-joining pathway. Although a shared feature of DNA ligases is their envelopment of the nicked duplex as a C-shaped protein clamp, they accomplish this feat by using remarkably different accessory structural modules and domain topologies. As structural, biochemical, and phylogenetic insights coalesce, we can expect advances on several fronts, including (i) pharmacological targeting of ligases for antibacterial and anticancer therapies and (ii) the discovery and design of new strand-sealing enzymes with unique substrate specificities.

 
 
 
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