This research examines the molecular events that occur when DNA polymerases, the enzymes responsible for duplicating DNA, encounter damage on DNA. In particular, the main question of this project is: what do DNA polymerases do when they encounter DNA strand breaks? The two strands of DNA in all living cells can be broken as a result of exposure to ionizing radiation or spontaneously through normal metabolic processes. DNA double-strand breaks are very harmful to a cell. Some estimates suggest one DNA double-strand break in a human cell is sufficient to lead to cell death. A DNA polymerase molecule is dependent on an unbroken DNA template for synthesis of new DNA; it uses the sequence of the template strand to create a new complementary copy of DNA. Therefore, one would expect the enzyme to simply stop or fall off when it reaches a DNA double-strand break. But this project focuses on a surprising observation: occasionally a DNA polymerase can synthesize DNA past a break in a template strand; it is able to switch between two broken template strands. The biological implications for DNA double-strand break repair and rejoining are important. DNA polymerases may play an important role in rejoining broken DNA templates during their replication of the genome. This project focuses on quantifying this template-switching activity by DNA polymerases and on determining the underlying mechanisms involved. Using a variety of experimental approaches from protein biochemistry to molecular biology, this research seeks to understand how DNA polymerases recognize broken template strands, what proteins influence template switching by DNA polymerases, and how DNA structure affects the efficiency of template switching.
