Response of the replisome to araA and araC suggests a mechanism for arabinosyl nucleoside tolerance.

Arabinosyl (ara) nucleoside analogues such as vidarabine (araA) and cytarabine (araC) are antimetabolites known to inhibit DNA polymerases in isolation. However, their effects on DNA synthesis catalysed by the multi-protein replisome remain unknown. Here, we uncover a central role for the lagging-strand DNA polymerase, Pol δ, in tolerating these drugs. Upon DNA synthesis inhibition by ara nucleotides, Pol δ replaces the canonical leading-strand DNA polymerase, Pol ε, to synthesise and proofread nascent leading strands in vitro and in vivo, with these Pol δ activities limiting helicase-polymerase uncoupling and intra-S checkpoint activation. Removal of ara analogues from nascent strands by DNA polymerase proofreading is likely the primary resistance mechanism against these drugs since a template-embedded analogue can be bypassed by the replisome. This study defines the initial response of the eukaryotic replisome to araA and araC and may have implications for how resistance arises against them, and other nucleoside analogue chemotherapeutics, in the clinic.