DNA replication fork stabilization and restart mechanisms revealed by biochemical reconstitution
Canal, B.; Bertolin, A. P.; Lee, G. C.; Minamino, M.; Diffley, J. F. X.
Show abstract
Understanding how DNA replication forks stall and restart and how the DNA damage checkpoint prevents irreversible fork collapse in molecular detail are crucial for understanding how cells maintain stable genomes and how they prevent the genetic instability that drives cancer. Here we describe the reconstitution of fork stalling and restart with purified budding yeast proteins. After nucleotide depletion, leading strand DNA synthesis quickly stops but CMG helicase continues to unwind and Okazaki fragments continue to initiate on the lagging strand. Incomplete Okazaki fragments sequester PCNA, RFC and DNA polymerases {delta} and {varepsilon} which prevents normal DNA synthesis restart and exposes nascent DNA to nuclease attack. The DNA damage checkpoint limits this sequestration by restraining fork progression, which protects stalled forks from collapse and ensures restart.
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