Efficient Extension of Slipped DNA Intermediates by DinB Is Required To Escape Primer Template Realignment by DnaQ

Abstract

We show that Escherichia coli DinB polymerase, which creates single-base deletions, prefers to extend slipped DNA substrates with the skipped base at the ؊4 position. A DinB(Y79L) variant, which extends these substrates less efficiently in vitro, allows the proofreading function of polymerase III to reverse their formation in vivo.The Y family of DNA polymerases catalyze replication on damaged DNA templates in a process called translesion DNA synthesis (TLS), which allows cells to tolerate DNA damage (7). Members of the Y family make relatively few contacts with the substrate DNA and incoming deoxynucleoside triphosphate (dNTP), which allows these enzymes to accommodate DNA lesions within their active sites (7). Several Y-family DNA polymerases have a marked preference for particular classes of DNA lesions (11,14,15), not only because of their open active sites but also due to their evolutionary divergent little-finger domains (for a review, see reference 17). In particular, DinB orthologs, which are the most ubiquitous of this polymerase class, are capable of bypassing certain N 2 -deoxyguanosine (N 2 -dG) adducts efficiently and accurately (9, 10). Escherichia coli DinB, the founding member of this subfamily, inserts dCTP across from N 2 -furfuryl-dG with 15-fold more catalytic efficiency than that found when dCTP is inserted across from an undamaged dG (10). DinB also extends from a dC:N 2 -furfuryl-dG base pair with a 25-fold increase in catalytic efficiency compared to a C ⅐ G base pair (9). However, the DinB variant DinB(Y79L) stalls 3 nucleotides downstream of the N 2 -furfuryl-dG lesion in vitro and, when expressed in vivo, results in death when cells are challenged with nitrofurazone, a compound which produces N 2 -dG adducts (9). Therefore, successful TLS requires not only insertion of the correct nucleotide opposite the lesion, but also, as suggested by our DinB(Y79L) results, efficient extension from it to avoid cell death (9).Due to their accommodating active sites and fewer enzyme-DNA contacts, DinB, and all TLS polymerases, have lower replication fidelity on undamaged DNA than replicative polymerases. We have recently shown that Escherichia coli DinB (Pol IV) creates Ϫ1 frameshifts via a template-slippage mechanism on homopolymeric nucleotide runs (6). The templateslippage mechanism, originally proposed by Streisinger et al. (18), suggests that the polymerase can misalign the primer and template strand, thereby resulting in an unpaired "extrahelical" base within the template strand (Fig. 1A). We proposed that DinB binds DNA templates containing homopolymeric nucleotide runs in a equilibrium between slipped and nonslipped DNA conformations, consistent with the template-slippage model (6). The incoming dNTP then displaces the equilibrium in favor of the slipped DNA conformation, thereby potentially resulting in a single-base deletion. However, it is still unclear which base within a homopolymeric run is most likely to become extrahelical during a DinB-mediated template-slippage reaction.In vitro pri...

Human polymerase kappa uses a template-slippage deletion mechanism, but can realign the slipped strands to favour base substitution mutations over deletions

“…An unpaired base at the −2 position is used ∼10-fold less efficiently than at the −3 position, with a k obs of 0.38 ± 0.04 s − 1 , suggesting that proximity to the active site reduces tolerance for an extrahelical nucleotide. This preference for an unpaired base further away from the active site has been observed for other DinB polymerases as well (27,28,30). …”

“…Having already ruled out misincorporation-misalignment for the 4C-G template, we used these altered substrates to distinguish template slippage from dNTP-stabilized misalignment. Similar DNA substrates have been used to assess deletion mechanisms for other DinB polymerases (27,28,30). …”

Translesion DNA Synthesis and Mutagenesis in Prokaryotes

“…In contrast, when the TLS patch is !5 nucleotides long, Pol III extends the primer and thus successfully completes the TLS pathway. Similarly, Walker and colleagues observed that efficient bypass of an N 2 -furfuryl-guanine adduct by Pol IV requires both insertion across the lesion and subsequent extension by at least four bases to prevent degradation by proofreading (Jarosz et al 2009;Foti and Walker 2011). It should thus be stressed that the most important factor that determines success or failure of a TLS pathway resides in the length of the TLS patch given that polymerase is able to synthesize under single-hit conditions.…”

A Polymerase – Tautomeric Model for Targeted Frameshift Mutations: Deletions Formation during Error-prone or SOS Replication of Double-stranded DNA Containing cis-syn Cyclobutane Thymine Dimers

“…DNA polymerase IV uses a template slippage mechanism to create single base deletions on homopolymeric runs [35]. DNA polymerase IV, which creates single-base deletions, prefers to extend slipped DNA substrates with the skipped base at the −4 position [36]. Overexpression of enzymes of the base excision repair pathway is known to increase the frequency of frameshift mutations [37].…”