Understanding the mechanisms of the enzymes catalyzing DNA replication is an important problem at the foundation of molecular biology. The collaborative coupling between helicase, DNA polymerase, and primase enzymes results in an exact copy of the genome. Replication errors are a major cause of genome instability and diseases such as cancer. For example, Bloom’s and Werner’s syndromes are associated with helicase defects, and mutations in mitochondrial helicase and polymerase cause mitochondrial DNA deletions associated with diseases such as premature ageing and neuromuscular disorders. A detailed understanding of the enzymatic mechanisms of helicase and polymerase mechanisms will enable development of therapeutics for such diseases.

Our studies focused on the replicative enzymes of bacteriophage T7 and human mitochondria. T7 system is also a model of human mitochondrial DNA replication since they share considerable similarity in amino acid sequence. In both systems, replication is reconstituted with three or four proteins, including a ring-shaped hexameric helicase (and primase in T7), DNA polymerase, and single-strand DNA binding protein.

Our current research on DNA replication addresses these key questions:

• How are the helicase and polymerase physically and functionally coupled at the replication fork?
• What is the structure of the replisome?
• What is the stepping mechanism of the replisome?
• How does the collaborative coupling affect DNA synthesis fidelity?
• What is the mechanism of coupling between leading and lagging strand DNA synthesis? 
• How do specific disease-related point mutations in the mitochondrial helicase Twinkle change its function compared to wild-type?

Funded by the NIGMS grant R35 118086: Mechanistic studies of nucleic acid enzymes involved in DNA replication, transcription, and innate immunity