Abstract
DNA2 is a nuclease/helicase that is involved in Okazaki fragment maturation, replication fork processing, and end resection of DNA double‐strand breaks. Similar such helicase activity for resolving secondary structures and structure‐specific nuclease activity are needed during DNA replication to process the chromosome‐specific higher order repeat units present in the centromeres of human chromosomes. Here, we show that DNA2 binds preferentially to centromeric DNA. The nuclease and helicase activities of DNA2 are both essential for resolution of DNA structural obstacles to facilitate DNA replication fork movement. Loss of DNA2‐mediated clean‐up mechanisms impairs centromeric DNA replication and CENP‐A deposition, leading to activation of the ATR DNA damage checkpoints at centromeric DNA regions and late‐S/G2 cell cycle arrest. Cells that escape arrest show impaired metaphase plate formation and abnormal chromosomal segregation. Furthermore, the DNA2 inhibitor C5 mimics DNA2 knockout and synergistically kills cancer cells when combined with an ATR inhibitor. These findings provide mechanistic insights into how DNA2 supports replication of centromeric DNA and give further insights into new therapeutic strategies.
Highlights
DNA2 is a nuclease/helicase that is involved in Okazaki fragment maturation, replication fork processing, and end resection of DNA double-strand breaks
In contrast to DNA2, when we used flap endonuclease 1 (FEN1) chromatin immunoprecipitation (ChIP) DNA as a template, qPCR did not show any preferential recruitment of FEN1 to the centromeric DNA (Figs 1B and EV1B), suggesting these cellular nucleases have differing functions during DNA replication
It is well known that a-satellite DNA in centromere regions is subject to secondary structures when the DNA replication machinery opens double-stranded DNA
Summary
DNA2 is a nuclease/helicase that is involved in Okazaki fragment maturation, replication fork processing, and end resection of DNA double-strand breaks. Similar such helicase activity for resolving secondary structures and structure-specific nuclease activity are needed during DNA replication to process the chromosome-specific higher order repeat units present in the centromeres of human chromosomes. The DNA2 inhibitor C5 mimics DNA2 knockout and synergistically kills cancer cells when combined with an ATR inhibitor. These findings provide mechanistic insights into how DNA2 supports replication of centromeric DNA and give further insights into new therapeutic strategies
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