Abstract
Translesion synthesis (TLS) by Y-family DNA polymerases alleviates replication stalling at DNA damage. Ring-shaped processivity clamps play a critical but ill-defined role in mediating exchange between Y-family and replicative polymerases during TLS. By reconstituting TLS at the single-molecule level, we show that the Escherichia coli β clamp can simultaneously bind the replicative polymerase (Pol) III and the conserved Y-family Pol IV, enabling exchange of the two polymerases and rapid bypass of a Pol IV cognate lesion. Furthermore, we find that a secondary contact between Pol IV and β limits Pol IV synthesis under normal conditions but facilitates Pol III displacement from the primer terminus following Pol IV induction during the SOS DNA damage response. These results support a role for secondary polymerase clamp interactions in regulating exchange and establishing a polymerase hierarchy.
Highlights
By occupying the rim and cleft sites of β in an inactive mode during normal growth conditions, Pol IV is available for rapid exchange and translesion synthesis when Pol III stalls on encountering a lesion, which was proposed in the toolbelt model [6]
We have shown that a previously unidentified inactive binding mode for Pol III allows it to remain bound to the cleft of one protomer of β until the switch back (Fig. 4 and Fig. S7A), the other half of the polymerase exchange reaction that has been difficult to resolve by bulk biochemical studies [3, 4]
We propose that this decrease is caused by an increased occupancy of Pol IV at the low-affinity rim sites of β (Fig. 6)
Summary
Single-Molecule Assay to Measure DNA Polymerase Activity. We used an assay that exploits the differential elasticity of ssDNA and dsDNA to observe primer extension on individual DNA molecules within a microfluidic flow cell [20, 21]. If the fraction of active protein differs for each one, the molar ratio of active polymerases will be shifted by a constant factor Under these conditions, Pol III performed 78% of DNA synthesis (Fig. S4), likely because of stronger interactions with β [2]; one or more Pol IV events were observed in 75% of trajectories (Fig. 2B), exchanging with Pol III. Previous studies have shown that Pol III lesion bypass efficiency is strongly promoted by β [28] and increased dNTP levels, which bias polymerase over exonuclease activity [29]; we, observed that higher dNTP levels increased bypass (Fig. S5B) The addition of both polymerases to the primer extension reaction alleviated the block at the N2furfuryl-dG position (Fig. 3A) and revealed polymerase exchange at the lesion site and bypass by Pol IV (Fig. 3B)
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