Defining the sequence requirements for the positioning of base J in DNA using SMRT sequencing

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• Base J (β-D-glucosyl-hydroxymethyluracil) is a unique DNA modification found in Leishmania.
• It replaces thymidine (T) and is primarily located in telomeric repeats and gene regulatory regions.
• The enzymes JBP1 and JBP2 are responsible for the synthesis of Base J.

Purpose of the Study:

• To identify the specific DNA sequences recognized by JBP1 and JBP2 for Base J synthesis.
• To elucidate the mechanism of Base J formation and maintenance in the Leishmania genome.

Main Methods:

• SMRT sequencing was employed to analyze DNA segments containing Base J.
• Plasmids with Leishmania DNA segments were grown in Leishmania tarentolae to study modification.
• Experiments were conducted using JBP2-null cells to assess enzyme function.

Main Results:

• SMRT sequencing successfully detected Base J modification in DNA.
• Leishmania DNA segments, including telomeric repeats, were modified when present in plasmids.
• Base J modification occurred at specific thymidine pairs on opposite DNA strands, near G-rich sequences.
• JBP2 was essential for de novo Base J insertion, while JBP1 maintained existing Base J.

Conclusions:

• JBP1 and JBP2 recognize specific DNA sequence motifs, including telomeric repeats and G-quadruplex forming regions, for Base J synthesis.
• A model is proposed where JBP2 mediates initial Base J insertion, and JBP1 maintains it post-replication.
• This study provides insights into the targeted DNA modification by JBP enzymes in Leishmania.