Virus-encoded glycosyltransferases hypermodify DNA with diverse glycans

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• Enzymatic modification of DNA nucleobases plays crucial roles in gene regulation and genome stability.
• Phage-specific enzymes, including cytosine methyltransferases (MT) and 5-methylpyrimidine dioxygenases (5mYOX), produce 5-hydroxymethylcytosine (5hmC) on viral DNA.
• 5hmC serves as a precursor for further enzymatic hypermodifications on viral genomes.

Purpose of the Study:

• To identify and characterize phage-associated glycosyltransferases (GTs) that modify 5hmC.
• To explore the diversity of sugar modifications on cytosine bases.
• To investigate the origins and biosynthetic pathways of enzymes involved in cytosine sugar hypermodification.

Main Methods:

• Metavirome mining to identify potential biosynthetic gene clusters for cytosine sugar hypermodification.
• Development of a high-throughput screening platform using the Escherichia coli metabolome.
• Reconstitution of enzymatic pathways and isolation of modified nucleobases.

Main Results:

• Identification of phage MT- and 5mYOX-associated GTs.
• Discovery of thousands of biosynthetic gene clusters involved in cytosine sugar hypermodification.
• Successful reconstitution of pathways yielding diverse sugar modifications on cytosine, including mono-, di-, and tri-saccharides (hexoses, N-acetylhexosamines, heptose).

Conclusions:

• Phage-associated GTs expand the known repertoire of nucleic acid hypermodifications.
• The study reveals novel enzymatic mechanisms for installing diverse sugars onto DNA bases.
• Findings contribute to understanding the evolution and diversity of DNA modification enzymes in viral systems.