Abstract
Cyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond. It was recently demonstrated that a homologue of adenylosuccinate synthetase (PurZ) and a dATP triphosphohydrolase (DatZ) are two important pieces of the metabolism of 2-aminoadenine, participating in the synthesis of ZTGC-DNA. Here, we determine that S-2L PurZ can use either dATP or ATP as a source of energy, thereby also depleting the pool of nucleotides in dATP. Furthermore, we identify a conserved gene (mazZ) located between purZ and datZ genes in S-2L and related phage genomes. We show that it encodes a (d)GTP-specific diphosphohydrolase, thereby providing the substrate of PurZ in the 2-aminoadenine synthesis pathway. High-resolution crystal structures of S-2L PurZ and MazZ with their respective substrates provide a rationale for their specificities. The Z-cluster made of these three genes – datZ, mazZ and purZ – was expressed in E. coli, resulting in a successful incorporation of 2-aminoadenine in the bacterial chromosomal and plasmidic DNA. This work opens the possibility to study synthetic organisms containing ZTGC-DNA.
Highlights
Cyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond
The S-2L genome can be divided into two parts, where almost all genes follow only one direction of translation; in-between, we identified a marRtype repressor found typically on such junctions[15]
On the day of submitting this paper, we became aware of the work of Zhou et al.[26] on the same subject
Summary
Cyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond. We identify a conserved gene (mazZ) located between purZ and datZ genes in S-2L and related phage genomes We show that it encodes a (d)GTP-specific diphosphohydrolase, thereby providing the substrate of PurZ in the 2-aminoadenine synthesis pathway. At least since the last universal common ancestor (LUCA), four types of nucleobases — adenine (A), guanine (G), cytosine (C) and thymine (T) — are used to encode genetic information in the DNA of all living cells[1]. Phage 9g replaces a quarter of its genomic guanine with archaeosine (G+)[6], enabling its DNA to resist 71% of cellular restriction enzymes[7] Such alterations are made almost exclusively without changing the Watson–Crick base-pairing scheme. The resulting ZTGC-DNA has an improved thermal stability[9,10] and proves to be almost completely resistant to adenine-targeting restriction enzymes[11]
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