Visualization of altered replication dynamics after DNA damage in human cells

Abstract

Eukaryotic cells respond to DNA damage within the S phase by activating an intra-S checkpoint: a response that includes reducing the rate of DNA synthesis. In yeast cells this can occur via checkpoint-dependent inhibition of origin firing and stabilization of ongoing forks, together with a checkpoint-independent slowing of fork movement. In higher eukaryotes, however, the mechanism by which DNA synthesis is reduced is less clear. We have developed strategies based on DNA fiber labeling that allow the quantitative assessment of rates of replication fork movement, origin firing, and fork stalling throughout the genome by examining large numbers of individually labeled replication forks. We show that exposing S phase cells to ionizing radiation induces a transient block to origin firing but does not affect fork rate or fork stalling. Alkylation damage by methyl methane sulfonate causes a slowing of fork movement and a high rate of fork stalling, in addition to inducing a block to new origin firing. Nucleotide depletion by hydroxyurea also reduces replication fork rate and increases stalling; moreover, in contrast to a recent report, we show that hydroxyurea induces a strong block to new origin firing. The DNA fiber labeling strategy provides a powerful new approach to analyze the dynamics of DNA replication in a perturbed S phase.