Abstract
DNA replication stress is often defined by the slowing or stalling of replication fork progression leading to local or global DNA synthesis inhibition. Failure to resolve replication stress in a timely manner contribute toward cell cycle defects, genome instability and human disease; however, the mechanism for fork recovery remains poorly defined. Here, we show that the translesion DNA polymerase (Pol) kappa, a DinB orthologue, has a unique role in both protecting and restarting stalled replication forks under conditions of nucleotide deprivation. Importantly, Pol kappa-mediated DNA synthesis during hydroxyurea (HU)-dependent fork restart is regulated by both the Fanconi Anemia (FA) pathway and PCNA polyubiquitination. Loss of Pol kappa prevents timely rescue of stalled replication forks, leading to replication-associated genomic instability, and a p53-dependent cell cycle defect. Taken together, our results identify a previously unanticipated role for Pol kappa in promoting DNA synthesis and replication stress recovery at sites of stalled forks.
Highlights
Replication stress in dividing cells often leads to increased stalled forks and under-replication of the DNA, which can contribute to elevated DNA damage, mutagenesis and genomic instability (Zeman and Cimprich, 2014)
To understand the molecular basis of individual DNA synthesis events under replication stress conditions, we employed the single-molecule DNA fiber technique to study the role of TLS Pols in modulating efficient fork restart from HU-induced stalled replication forks (Figure 1A)
One such barrier involves oncogene-induced DNA replication stress, which leads to activation of the DNA damage checkpoint, cell cycle arrest and/or senescence and apoptosis (Schoppy et al, 2012; Gilad et al, 2010; Murga et al, 2011)
Summary
Replication stress in dividing cells often leads to increased stalled forks and under-replication of the DNA, which can contribute to elevated DNA damage, mutagenesis and genomic instability (Zeman and Cimprich, 2014). Stalled replication forks that are stabilized by the ATR pathway can be restarted by these error-prone TLS Pols when the source of the stress itself (including bulky DNA adducts or UV crosslinks) cannot be removed in a timely fashion, as in the case of an unrepaired DNA lesion (Zeman and Cimprich, 2014). We found that the TLS Pol kappa (PolK) plays a critical role in fork restart during high-dose HU (2 mM) treatment Both the FA pathway and PCNA polyubiquitination are critical regulators of PolK recruitment and function at stalled forks. These findings reveal a previously unappreciated role for a Y-family TLS Pol in promoting replication fork recovery during conditions of nucleotide deprivation
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