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

Abstract

During DNA replication, the presence of 8-oxoguanine (8-oxoG) lesions in the template strand cause the high-fidelity (HiFi) DNA polymerase (Pol) to stall. An early response to 8-oxoG lesions involves 'on-the-fly' translesion synthesis (TLS), in which a specialized TLS Pol is recruited and replaces the stalled HiFi Pol for lesion bypass. The length of TLS must be long enough for effective bypass, but it must also be regulated to minimize replication errors by the TLS Pol. The exact position where the TLS Pol ends and the HiFi Pol resumes (i.e. the length of the TLS patch) has not been described. We use steady-state and pre-steady-state kinetic assays to characterize lesion bypass intermediates formed by different archaeal polymerase holoenzyme complexes that include PCNA123 and RFC. After bypass of 8-oxoG by TLS PolY, products accumulate at the template position three base pairs beyond the lesion. PolY is catalytically poor for subsequent extension from this +3 position beyond 8-oxoG, but this inefficiency is overcome by rapid extension of HiFi PolB1. The reciprocation of Pol activities at this intermediate indicates a defined position where TLS Pol extension is limited and where the DNA substrate is handed back to the HiFi Pol after bypass of 8-oxoG.

Figure 1.

PolY and PolB1 have complementing activities for bypass and extension from an 8-oxoG lesion. (A) A 52-mer DNA template containing an 8-oxoG lesion at position 23 (from the 3′ end) was annealed to primers of progressing lengths. DNA sequences for each oligo are listed in Supplemental Table S1. Steady-state lesion bypass assays were performed by pre-loading the indicated DNA substrate with different replication subassemblies at indicated (final) concentrations (see Materials and Methods). Lesion bypass reactions performed on (B) the running start substrate and (C) the stalled substrates were resolved by low-resolution denaturing PAGE. The ‘?’ highlights an intermediate product of undefined length formed by YHE downstream of 8-oxoG. Products formed by the indicated Pol complex were quantified for percentage of ‘Bypassed’ products beyond the lesion position. (D) Steady-state extension assays were performed on substrates with longer starting primers. Products quantified for the percentage of primer that was utilized by each Pol complex (B1HE, purple -•-; YHE, red -▪-; SHE, blue -♦-). Gel images are in Supplemental Figure S2. FL indicates the position of full-length product, and arrows indicate an intermediate product downstream of 8-oxoG. Error bars represent the standard deviation from three independent replicates.

Figure 2.

Lesion bypass by PolY yields a major intermediate 3 bp downstream of the 8-oxoG lesion. (A) Representative lesion bypass and extension products by different Pol complexes (from Figure 1 and Supplemental Figure S2) were resolved by high-resolution denaturing PAGE. The arrow indicates the intermediate product 3 base pairs beyond (+3) the 8-oxoG lesion, and ‘Extended’ indicates all products beyond the +3 position. The normalized fraction of (B) ‘+3’ product bands and (C) ‘Extended’ products for the YHE (red) or SHE (blue) were plotted from indicated primer starting positions.

Figure 3.

Kinetics of lesion bypass by the YHE complexes demonstrate accumulation of the +3 intermediate. (A) Pre-steady-state lesion bypass assays were performed on substrates containing the stalled primer, p22 (−1), by Schemes i (red) and ii (orange). (B) Products were resolved by denaturing PAGE and (C) quantified for ‘Total Bypass’ (black -•-, solid line fit to Equation 1), or ‘0 to +2’ (-▪-, smoothed solid line), ‘+3’ (-○-, smoothed dashed line), and ‘Extended’ (-▴-, smoothed solid line) intermediates. Error bars represent standard deviation from three independent replicates of each time point.

Figure 4.

Kinetics of lesion bypass by the SHE complexes yield less +3 intermediate. Pre-steady-state lesion bypass assays were performed by (A) Scheme iii (yellow) and (B) Scheme iv (green). Products by Schemes iii & iv were quantified for ‘Total Bypass’ (black -•-, solid line fit to Equation 1) and intermediate products ‘0 to +2’ (-▪-, smoothed solid trace), ‘+3’ (-○-, smoothed dashed trace) and ‘Extended’ (-▴-, smoothed solid trace) were traced. Error bars represent standard deviation from three independent replicates of each time point. (C) The observed rate constants of lesion bypass for Schemes i–iv were plotted (colors correspond to the respective schemes). Error bars indicate error of the regression fit. (D) The maximal fraction of the ‘+3’ intermediate products from lesion bypass of Schemes i–iv (dashed traces) were plotted (colors correspond to the respective schemes). Error bars represent standard deviation from three replicates for each scheme.

Figure 5.

PolY conducts slow extension from the +3 intermediate. (A) Pre-steady-state extension assays were performed on DNA substrates containing primer p26 (+3) by Schemes i & ii. Quantification of ‘Extended’ products were fit to (Equation 1) to obtain the observed rate constants for Scheme i (red, -•-) and ii (orange, -▴-). (B) Single nucleotide extension assays were performed with increasing concentrations of dATP by Scheme i on an undamaged or (C) and 8-oxoG damaged template from the p26 (+3) primer as indicated in the legends. Quantification of ‘Extended’ products were fit to Equation (1) to obtain the observed rate constants at indicated concentrations of dATP. Second order plots (insets) report on the kinetics of dATP insertion at +3 on undamaged (0.92 ± 0.01 nM⁻¹s⁻¹) or 8-oxoG damaged (0.43 ± 0.01 nM⁻¹s⁻¹) templates.

Figure 6.

PolB1 conducts rapid extension from the +3 intermediate position. Pre-steady-state extension kinetics were performed on a DNA substrate containing primer p26 (+3) by (A) Schemes iii (yellow, -•-) and iv (green, -▴-) containing PolB1 and PolY, or (B) Schemes v (blue, -•-) and vi (purple, -▴-) containing PolB1 alone. Error bars represent standard deviation from three independent replicates of each time point. Quantification of ‘Extended’ products were fit to Equation (1) to obtain the observed rate constant for extension. Insets show the trace of ‘Extended’ product at a shorter time scale. (C) Observed rate constants for extension from +3 were plotted; error bars indicate error of the regression fit.

Figure 7.

Proposed mechanism for DNA hand-offs between PolB1 and PolY in response to an 8-oxoG lesion. (A) Upon encountering 8-oxoG on the template strand, PolB1 stalls. (B) Individual contacts with PCNA123 mediate the first Pol hand-off from PolB1 to PolY. (C) PolY then performs translesion synthesis to the template position three base pairs beyond the 8-oxoG lesion, where it becomes catalytically inefficient for additional synthesis. (D) The second Pol hand-off from PolY to PolB1 is then mediated through PCNA123 to re-establish HiFi PolB1 for (E) extension and effective bypass of the lesion. The shaded step highlights the second transient polymerase handoff to restore high fidelity synthesis.