Dynamics of replication origin over-activation

Abstract

We determined replication patterns in cancer cells in which the controls that normally prevent excess replication were disrupted ("re-replicating cells"). Single-fiber analyses suggested that replication origins were activated at a higher frequency in re-replicating cells. However, nascent strand sequencing demonstrated that re-replicating cells utilized the same pool of potential replication origins as normally replicating cells. Surprisingly, rereplicating cells exhibited a skewed initiation frequency correlating with replication timing.These patterns differed from the replication profiles observed in non-re-replicating cells exposed to replication stress, which activated a novel group of dormant origins not typically activated during normal mitotic growth. Hence, disruption of the molecular interactions that regulates origin initiation can activate two distinct pools of potential replication origins: rereplicating cells over-activate flexible origins while replication stress in normal mitotic growth activates dormant origins.Our previous studies have shown that RepID, a replicator-specific binding protein essential for site-specific initiation of DNA replication in mammalian cells 25 , recruits CRL4 to chromatin in a PCNA-independent manner prior to the onset of S phase and promotes CDT1 degradation.RepID deficiency is associated with delayed CDT1 degradation, resulting in limited genome rereplication 26 . Partial genome re-replication can also be caused by inhibition of DOT1L, a methyltransferase which catalyzes the demethylation of histone H3 on lysine 79 (H3K79Me2), thereby removing a histone modification typically associated with a group of replication origins 27 . Because massive re-replication can drive cell death specifically in checkpointcompromised cancer cells, both CDT1 stabilization by inactivation of ubiquitin-mediated degradation and inhibition of DOT1L are currently being explored as novel anti-cancer therapeutic strategies [28][29][30][31][32][33] .Given that genome re-replication is a common avenue to genomic instability and considering its potential as a strategy for chemotherapy, it is important to understand in detail its mechanics and downstream consequences. Here, we report that re-replication exhibits aberrant replication fork dynamics and occurs more slowly than the routine genome duplication that takes place during normal growth. The consequences of the persistent presence of pre-replication complexes on chromatin include massive DNA damage and the induction of senescence. Unlike other instances of replication origin over-activation, such as when replication slows down as a result of nucleotide depletion or in cells exposed to DNA damaging conditions, the re-replication process preferentially utilizes a subset of the replication origin pool typically used during normal growth. Results: