A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

Abstract

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability.Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the bacterial replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of TLS polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, and the concomitant increase in TLS polymerase levels, higher fractions of stalled replisomes are resolved through TLS (Fuchs, 2016;.

Visualizing mutagenic repair: novel insights into bacterial translesion synthesis

“…This binding is critical for the gate-keeping function of ε to regulate promiscuous access of β-clamp by Pol IV (Chang et al . 2019 ). Levels of Pol IV would then dictate whether the TLS polymerase gains access to the replisome at the site of the lesion itself or if the replisome would skip the lesion and TLS would then occur behind the replication fork.…”

“…This is supported by a recent study that suggests that the gate-keeping function of ε determines the pathway choice between TLS at and behind the fork (Chang et al . 2019 ).…”

“…It is probable that these are competing mechanisms; a combination of both occurs, possibly dictated by the levels of the TLS polymerase at a given instance (Gabbai, Yeeles and Marians 2014 ; Chang et al . 2019 ). Apart from this, efficiency with which the polymerase can bypass different types of lesions would also have a significant impact on the mechanism of repair (Thrall et al .…”

“… 2016 ; Vaisman and Woodgate 2017 ; Yang and Gao 2018 ; Chang et al . 2019 ). The types and frequency of mutations vary based on the class of TLS polymerase as well as the type of DNA lesion the polymerase bypasses (summarized in Table 1 ).…”

A hand-off of DNA between archaeal polymerases allows high-fidelity replication to resume at a discrete intermediate three bases past 8-oxoguanine

“…Alternatively, TLS may occur 'on-the-fly,' in which a TLS Pol is recruited directly to a stalled replication fork to perform lesion bypass prior to a re-priming event (7). Recent in vitro studies have suggested that this continuous 'on-the-fly' TLS pathway is an early response to replication stalling lesions, whereas re-priming serves as a slower mechanism which is likely utilized during conditions of excessive DNA damage (8)(9)(10).…”