DNA Sequence Context Controls the Binding and Processivity of the T7 DNA Primase

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Primases are essential enzymes for DNA replication, synthesizing RNA primers for DNA polymerases.
• Understanding primase function, particularly DNA binding and activity, is crucial for comprehending replication mechanisms.

Purpose of the Study:

• To investigate the DNA binding and activity of bacteriophage T7 primase.
• To explore the impact of flanking DNA sequences on T7 primase recognition and function using a novel workflow.

Main Methods:

• Development and application of a high-throughput primase profiling (HTPP) workflow.
• Combination of high-throughput binding assays and biochemical analyses to assess primase-DNA interactions and activity.

Main Results:

• HTPP revealed complex binding specificity and functional activity patterns for T7 primase.
• G/T-rich sequences flanking the primase recognition site increased DNA binding up to 10-fold.
• These flanking sequences also unexpectedly increased RNA primer length by up to 3-fold, a previously unreported observation for T7 primase.

Conclusions:

• Flanking DNA sequences significantly modulate bacteriophage T7 primase binding and primer synthesis activity.
• The discovery of sequence-dependent primer length variability opens new avenues for understanding primase regulation.
• The HTPP workflow is a powerful tool for uncovering sequence-specific effects on DNA-interacting enzymes.