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, 112 (7), E633-8

Archaeal Replicative Primases Can Perform Translesion DNA Synthesis

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Archaeal Replicative Primases Can Perform Translesion DNA Synthesis

Stanislaw K Jozwiakowski et al. Proc Natl Acad Sci U S A.

Abstract

DNA replicases routinely stall at lesions encountered on the template strand, and translesion DNA synthesis (TLS) is used to rescue progression of stalled replisomes. This process requires specialized polymerases that perform translesion DNA synthesis. Although prokaryotes and eukaryotes possess canonical TLS polymerases (Y-family Pols) capable of traversing blocking DNA lesions, most archaea lack these enzymes. Here, we report that archaeal replicative primases (Pri S, primase small subunit) can also perform TLS. Archaeal Pri S can bypass common oxidative DNA lesions, such as 8-Oxo-2'-deoxyguanosines and UV light-induced DNA damage, faithfully bypassing cyclobutane pyrimidine dimers. Although it is well documented that archaeal replicases specifically arrest at deoxyuracils (dUs) due to recognition and binding to the lesions, a replication restart mechanism has not been identified. Here, we report that Pri S efficiently replicates past dUs, even in the presence of stalled replicase complexes, thus providing a mechanism for maintaining replication bypass of these DNA lesions. Together, these findings establish that some replicative primases, previously considered to be solely involved in priming replication, are also TLS proficient and therefore may play important roles in damage tolerance at replication forks.

Keywords: AEP; archaea; primase; replication; translesion synthesis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
A. fulgidus replisomal enzymes displaying DNA polymerase activity. Analysis of 173 archaeal genomes revealed that only 79 archaea encode canonical TLS DNA polymerases. DNA polymerization activities of A. fulgidus replisomal enzymes. (A) The absence of genes encoding Y-family DNA polymerases in most archaea is shown. (B) Structural elements present in A. fulgidus replisomal enzymes. Abbreviations: AEP, archaeo-eukaryotic primase; CTD, carboxy terminal domain; Exo, exonuclease; NTD, amino terminal domain; Pol, polymerase; and Zn, zinc binding site. (C) Polymerization on nondamaged templates. (D) Single nucleotide incorporation on nondamaged templates. The letter C denotes no enzyme control. The triangles above gel panels indicate time course of the polymerization (30 s, 1', 5', and 10').
Fig. 2.
Fig. 2.
Translesion synthesis past 8-oxo-dGs, dUs, and CPDs. (A) TLS performed on templates containing 8-oxo-dGs. (B) Single nucleotide incorporation opposite 8-oxo-dG. (C) TLS performed on templates containing dUs. (D) Single nucleotide incorporation opposite dU. (E) TLS performed on templates containing CPDs. (F) Single nucleotide incorporation opposite CPD. The letter C denotes no enzyme control. The triangles above gel panels indicate time course of the polymerization (30 s, 1', 5', and 10').
Fig. 3.
Fig. 3.
Translesion synthesis past multiple DNA damage. (A) Pol B/PCNA stalls four bases before dU (Left). Stalled Pol B/PCNA is rescued when TLS past dU is performed by Pri S/L (Right). (B) Control primer extension on nondamaged substrates. Polymerization performed by Pol B (Left), Pol B/PCNA (Middle), and Pol B/PCNA and Pri S/L (Right). (C) Primer extension on templates containing seven 8-oxo dGs. TLS performed by Pol B and Pol B/PCNA results in full-length product (Left and Middle, respectively) but pronounced pausing pattern opposite to 8-oxo dG is observed. Polymerization performed by Pol B/PCNA and Pri S/L results in efficient primer extension. (D) Polymerization on a DNA template containing seven dUs performed by Pol B and Pol B/PCNA is strongly inhibited (Left and Middle, respectively). In contrast, DNA synthesis performed by Pol B/PCNA and Pri S/L resulted in bypass of the multiple dUs. (E) Primer extension on long nondamaged templates. Polymerization was performed by Pol B (Left), Pol B/PCNA (Middle), Pol B/PCNA and Pri S/L (Right). (F) Polymerization on long templates containing ∼20 randomly distributed dUs. Strong inhibition of DNA synthesis is observed for Pol B and Pol B/PCNA (Left and Middle, respectively). Again, DNA synthesis performed by Pol B/PCNA and Pri S/L resulted in full-length primer extension. The letter C denotes no enzyme control. The triangles above gel panels indicate time course of the polymerization; A (30 s, 1', 5', and 10'), B and C (1', 5', and 10'), and E and F (3', 10', and 20').
Fig. 4.
Fig. 4.
Collaboration of the core components of the archaeal replisome results in bypass of DNA lesions. Top shows polymerizing replicase (Pol B, blue) with sliding clamp (PCNA, gray). The complex encounters blocking DNA lesion (red triangle) resulting in Pol B idling, which allows recruitment of the primase (Pri S/L, yellow). Depending on the type of the lesion, Pri S/L employs either primase or translesion synthesis (TLS) activity. Left illustrates a scenario where the blocking lesion is relatively large (i.e., large aromatic organic compound or protein covalently attached to DNA) and Pri S/L synthesizes a short primer (orange) after the damage so that the Pol B/PCNA complex can restart replication downstream from the block. Alternatively, when the blocking lesion is small (e.g., 8-oxo dG, dU, or CPD), TLS (green) is performed by Pri S/L so that the Pol B/PCNA complex resumes DNA synthesis. The fidelity of TLS performed by Afu-Pri S/L is shown.

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