Distinct Phage‐Encoded Enzymes for Substitution of Deoxythymidine by Deoxyuridine in Phage Genomes

DNA base modification is a common strategy used by bacteriophages to evade host immune detection. A prominent example is dU‐DNA, where thymidine is globally replaced with 2′‐deoxyuridine. Despite its widespread occurrence, dU‐DNA's biosynthetic pathways and functional roles remain incompletely understood. Here, different enzymes supporting dU‐DNA biosynthesis in phage PBS1, Roseophage DSS3_VP1, and Yersiniophage PhiR1‐37 are identified and characterized. The dU nucleotide precursor is supplied by phage dCTP deaminases (Dcds). Thymidine nucleotides are degraded by dTMP phosphatases (Dtms) in PBS1 and DSS3_VP1, and by a dTTP pyrophosphatase (Dtt) in PhiR1‐37, preventing incorporation into phage DNA. The dU‐DNA, isolated from Yersiniophage PhiR1‐37 or synthesized by PCR, demonstrates resistance to cleavage by restriction enzymes recognizing thymidine‐containing sequences, partial resistance to Lb Cas12a nuclease recognizing a TTTV PAM site, while remaining sensitive to Sp Cas9 nuclease recognizing a NGG PAM site. A phylogenetic analysis of PBS1 dCTP deaminase and closely related T4 phage dCMP deaminase suggest possible evolutionary origins from bacterial dCDP deaminases. Overall, these findings suggest independent acquisition of dU‐DNA biosynthetic enzymes and pathways in the diverse phages, and support its protective function against different host‐encoded nucleases.