Mechanism of ultraviolet-induced mutagenesis: Extent and fidelity of in vitro DNA synthesis on irradiated templates

Abstract

The effect of UV irradiation on the extent and fidelity of DNA synthesis in vitro was studied by using homopolymers and primed single-stranded kX174 phage DNA polymerase I on irradiated 4X174 DNA has been observed. We propose that this nucleotide turnover is due to idling by DNA polymerase (i.e., incorporation and subsequent excision of nucleotides opposite UV photolesions, by the 3'_-5' "proofreading" exonuclease) thus preventing replication past pyrimidine dimers and the potentially mutagenic event that should result. In support of this hypothesis, DNA synthesis by DNA polymerase from avian myeloblastosis virus and by mammalian DNA polymerase a, both of which are devoid of any exonuclease activity, was found to be only partially inhibited, but not blocked, by UV irradiation of the template and accompanied by an increased incorporation of noncomplementary nucleotides. It is suggested that UV mutagenesis inbacteria requires an induced modification of the cellular DNA replication machinery, possibly an inhibition of the 3'-5' exonuclease activity associated with DNA polymerases.UV irradiation of X and 4X174 bacteriophages results in killing, but not mutagenesis, of phage after infection of untreated host cells (1, 2), whereas UV irradiation of the host cells causes mutagenesis of both untreated and irradiated phage (1-4).Furthermore, UV-irradiated single-stranded qX174 DNA extracted from untreated host cells was found to be replicated only to the first pyrimidine dimer (5, 6). Caillet-Fauquet et al. (6) found that UV irradiation of the host cell leads to an enhancement of the DNA synthesis on UV-irradiated OX174 DNA in vivo, which led them to propose that pyrimidine dimers in the cX174 DNA can become mutagenic by causing misincorporation of deoxyribonucleotides opposite them.This inducible mutagenic system is thought to be part of a complex cellular emergency response ("SOS induction") that is triggered by unrepaired DNA lesions (such as pyrimidine dimers). The response also includes the arrest of cellular division, the arrest of aerobic metabolism, and the induction of prophage in lysogenic bacteria (7,8).DNA polymerases (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) are known to be able to control the fidelity of DNA synthesis by selection of the correct nucleotide (9) and by possession of a "proof-reading" 3'-"5' exonuclease activity that can detect and excise 3'-terminal mismatched bases (10, 11).We have sought to understand the molecular mechanisms underlying UV-induced mutagenesis by an examination of the role of DNA

Levels of epsilon, an essential replication subunit of Escherichia coli DNA polymerase III, are controlled by heat shock proteins

“…Both in vivo and in vitro experiments indicate that insertion of a base opposite a lesion is not rate limiting but that extension from the mispaired terminus is (6,18). The two modifications of Pol III that appear to be required are inhibition of the 3'-*5' exonuclease activity of epsilon to prevent the complex from stalling at the mispaired terminus (54) and improving the ability of the polymerase to extend from the mispaired terminus (2,6,18). A number of lines of evidence support the idea that RecA and UmuDC are involved in performing these alterations: (i) overproduction * Corresponding author.…”

New mutations in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activity

“…In addition, it has been proposed that the RecA protein possesses at least one other role in mutagenesis (14,33). This could be either direct participation in the modified SOS replisome allowing the misincorporation step (27) or inhibition of the proofreading activity of DNA polymerase III (28,43). RecA* may also be required to activate another protein or inactivate another repressor (6).…”

Capacity of RecA protein to bind preferentially to UV lesions and inhibit the editing subunit (epsilon) of DNA polymerase III: a possible mechanism for SOS-induced targeted mutagenesis.

“…One suggested error mechanism is an inhibition of the editing capacity (3' --5' exonuclease) of the replicating DNA polymerase, allowing replication past the dimer with random base insertion (16). A modification of this proposal, specialized to RecA, is that RecA recognizes the DNA lesion and locally modifies the DNA polymerase III (polIII) holoenzyme at the dimer site to a reduced fidelity form; the enzyme then replicates through the dimer site, using the residual base-pairing capacity of the dimerized bases in spite of their distorted configuration (a localized relaxed-specificity mechanism) (17).…”