Replication Bypass of the Acrolein-Mediated Deoxyguanine DNA-Peptide Cross-Links by DNA Polymerases of the DinB Family

Abstract

DNA–protein cross-links (adducts) are formed in cellular DNA under a variety of conditions, particularly following exposure to an α,β-unsaturated aldehyde, acrolein. DNA–protein cross-links are subject to repair or damage-tolerance processes. These adducts serve as substrates for proteolytic degradation, yielding DNA–peptide lesions that have been shown to be actively repaired by the nucleotide excision repair complex. Alternatively, DNA–peptide cross-links can be subjected to replication bypass. We present new evidence about the capabilities of DNA polymerases to synthesize DNA past such cross-links. DNAs were constructed with site-specific cross-links, in which either a tetrapeptide or dodecylpeptide was covalently attached at the N2 position of guanine via an acrolein adduct, and replication bypass assays carried out with members of the DinB family of polymerases, human polymerase (pol)κ and Escherichia coli (E. coli) pol IV, and various E. coli polymerases that do not belong to the DinB family. Pol κ was able to catalyze both the incorporation and extension steps with an efficiency that was qualitatively indistinguishable from control (undamaged) substrates. Fidelity was comparable on all these substrates, suggesting that pol κ would have a role in the low mutation frequency associated with replication of these adducts in mammalian cells. When the E. coli orthologue of pol κ, damage-inducible DNA polymerase, pol IV, was analyzed on the same substrates, pause sites were detected opposite and three nucleotides beyond the site of the lesion, with incorporation opposite the lesion being accurate. In contrast, neither E. coli replicative polymerase, pol III, nor E. coli damage-inducible polymerases, pol II and pol V, could efficiently incorporate a nucleotide opposite the DNA–peptide cross-links. Consistent with a role for pol IV in tolerance of these lesions, the replication efficiency of DNAs containing DNA–peptide cross-links was greatly reduced in pol IV-deficient cells. Collectively, these data indicate an important role for the DinB family of polymerases in tolerance mechanisms of N2-guanine linked DNA–peptide cross-links.

Error-prone Translesion Synthesis Past DNA-Peptide Cross-links Conjugated to the Major Groove of DNA via C5 of Thymidine

“…Previous investigations have revealed that the ability of DNA polymerases to bypass DNA-peptide conjugates is dependent on the lesion size, the attachment site within the DNA, and polymerase identity (10,(21)(22)(23)(24)(25)(26)(27)(28)(29). Lloyd and co-workers (26) have reported that human polymerase (hpol) and its Escherichia coli orthologue pol IV were able to catalyze error-free primer extension past DNA templates containing tetra-and dodecapeptides conjugated to the N 2 position of guanine via a trimethylene linker.…”

“…Lloyd and co-workers (26) have reported that human polymerase (hpol) and its Escherichia coli orthologue pol IV were able to catalyze error-free primer extension past DNA templates containing tetra-and dodecapeptides conjugated to the N 2 position of guanine via a trimethylene linker. hpol was capable of bypassing both 4-and 12-mer peptides conjugated to N 6 of adenine via a trimethylene linkage, with correct base (dT) inserted opposite the adduct (27).…”

“…In the resulting primer-template complexes, the 3Ј-primer terminus is positioned immediately prior to the lesion site (Ϫ1 primer, Scheme 2A). Initial single nucleotide incorporation assays were conducted to determine what nucleotides can be inserted opposite the adducted site by human translesion synthesis polymerases, hpol and , which were selected due to their documented ability to catalyze replication past other bulky DNA lesions (26,29). The resulting primer-template duplexes were incubated with recombinant polymerases in the presence of individual dNTPs for 0 -90 min.…”

Roles for the transcription elongation factor NusA in both DNA repair and damage tolerance pathways in Escherichia coli

“…ΔdinB strains are sensitive to DNA-damaging agents, nitrofurazone (NFZ) and 4-nitroquinolone-1-oxide (4-NQO), and DinB preferentially and accurately bypasses a structural analog of the major NFZ-induced N 2 -dG lesion as well as certain other N 2 -dG adducts (10)(11)(12)(13). NusA is an essential, multidomain protein that functions in both termination and antitermination of transcription, and is associated with the RNA polymerase (RNAP) throughout the elongation and termination phases of transcription (14)(15)(16)(17)(18)(19)(20)(21)(22).…”

Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links

“…For example, treatment with a cytidine analog, azacytidine C, results in covalently linked methyltransferase-DNA adducts that cause blockage of DNA replication in vivo (49). This blockage may be overcome by the action of specialized, translesion synthesis DNA polymerases that have been shown to be required for bypassing DNA-peptide cross-links, the potential intermediates generated during processing of DPCs (50). A similar role can be predicted for the helicases, rationalizing for the existence of several specialized helicases that participate in processing of blocked replication forks via different repair processes in a lesion-specific manner.…”