A Biosynthetic Pathway For Substitution Of Adenine By Diaminopurine In Phage Genomes

DNA modifications vary in forms and physiological functions,and they generally do not alter Watson-Crick base pairing.The only exception is the 2,6-diaminopurine(Z)in the genomic DNA of cyanophage S-2L: Z completely replaces adenine(A)and pairs with thymine(T)forming three hydrogen bonds,which alter the physical and chemical properties of the DNA.For nearly half a century,the biosynthesis,prevalence and significance of the Z-genome still remain unexplored.A combination of gene annotation,enzyme sequence similarity network and genome neighborhood network analysis prompted us to hypothesize the involvement of multiple enzymes in the biosynthesis of the phage Z-genome.Among them,phage-encoded 2-aminodeoxyadenosylsuccinate synthetase(Pur Z)and two nucleotide hydrolyses(d ATPase and DUF550)play a critical role,demonstrated by in vitro enzyme activity assays,including enzyme coupling colorimetric assay and the detection of products by ultraviolet spectrometry and LC-MS.Phage encoded Pur Z catalyzing the ATP-dependent condensation of d GMP with aspartic acid to produce 2-aminodeoxyadenosylsuccinate,which is subsequently converted to d ZMP by adenylosuccinate lyase(Pur B).The d ZMP could then be phosphorylated by the host-encoded guanosine monophosphate kinase to form d ZTP for the incorporation into the phage genome.The nucleotide hydrolyses d ATPase and DUF550 were discovered on the gene cluster of Pur Z-containing phages.Phage encoded d ATPase was found to be a specific d ATP triphosphohydrolase,which removes d ATP from the nucleotide pool of phage-infected cells,preventing adenine from entering the phage’s genomic DNA.This study further characterized a phage-encoded enzyme,DUF550 containing unknown functional domain and demonstrated its d GTP/d ATP pyrophosphatase activity.The function of DUF550 is believed to further degrade the d ATP and increase the intracellular d GMP concentration,providing the physiological substrate for Pur Z,facilitating the biosynthesis of the phage Z-genome.Enzyme activity assays following site-directed mutagenesis identified the key residues in the Pur Z active site.With information of conserved amino acid residues,we identified hundreds of Pur Zs in both Siphoviridae and Podoviridae,widely distributed on Earth,and further verified the Z-genome in one of these phages,Acinetobacter phage SH-Ab 15497.Only the d Z,d G,d C and d T were detected in the enzymatic hydrolysis products of this phage genomic DNA,and the molar ratio of d Z:d T and d G:d C was close to 1:1.Therefore the Z-genome is not restricted in cyanophage S-2L,rather it is a general genomic format used by many phages.The Z-genome was further shown resistant to the digestions of restriction endonucleases with recognition sites containing one or more adenines,suggesting that harboring a Z-genome is a common strategy for phages to evade the host defense and thus have an evolutionary advantage.In addition,the Pur Z variant,Pur Z2 is also reported in this thesis.Pur Z2 differs from Pur Z in that it catalyzes the condensation of d GMP with aspartic acid in a GTP-dependent manner,whereas the Pur Z-catalyzed reaction is ATP-dependent.Both Pur Z2 and the ancient adenosylsuccinate synthetase(Pur A)use GTP as a substrate.Such commonality may underline further study of the Z-genome evolution.In summary,investigation of the biosynthesis and formation mechanism of phages Z-genome,this study firstly revealed for an important natural phenomenon and scientific laws that phages gain an evolutionary advantage by encoding Z-genome to evade host defense.Engineered lives equipped with the Z-genome biosynthetic machinery reported in this thesis may eventually allow the production of Z-base DNAs in large scale,advancing their applications in DNA origami,information storage,phage therapy,food preservation,and biological controls etc.