MECHANISTIC STUDIES OF REPLICATION FORK REMODELING BY DNA TRANSLOCASES

Abstract

During DNA replication the replication fork can become stalled by impediments that prevent complete and accurate duplication of the genome. DNA replication fork reversal is an important pathway for the protection and restoration of stalled DNA replication forks in both prokaryotes and eukaryotes to preserve genome stability. Several enzymes known as fork remodelers initiate this pathway by actively reversing a stalled DNA replication fork, although their mechanisms of action are not well understood. This dissertation presents work characterizing fork reversal mechanisms of the prokaryotic fork remodeler RecG and the eukaryotic SNF2-family fork remodelers SMARCAL1, ZRANB3, and HLTF. Biophysical analysis of RecG binding to a model DNA replication fork revealed how conformational changes within the motor domain upon binding DNA is required for efficient fork reversal activity. Comparison of fork reversal mechanisms of the eukaryotic fork remodelers show differences in sensitivity to DNA damage on the leading and lagging strand template, providing a rationale for the multiple non-redundant fork remodeling activities in the cell. This work has expanded our knowledge of how these enzymes recognize and remodel stalled replication forks to ensure genome stability.