Crosstalk between repair pathways elicits double-strand breaks in alkylated DNA and implications for the action of temozolomide.

Temozolomide (TMZ), a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O6-methylguanine (O6mG) adducts. Current models propose that during DNA replication, thymine is incorporated across from O6mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double-strand breaks (DSBs). To revisit the mechanism of O6mG processing, we reacted plasmid DNA with N-methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg-derived extracts. We have shown that in this system, MMR proteins are enriched on MNU-treated DNA and we observed robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O6mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increases linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O6mG:C sites and the other involving base excision repair acting at a nearby N-alkylation adduct. We propose a new, replication-independent mechanism of action of TMZ, which operates in addition to the well-studied cell cycle-dependent mode of action.

Enrichment of plasmid pool from E. coli for mass sequencing to detect untargeted mutagenic events.

Translesion synthesis (TLS) is an event to cope with DNA damages. During TLS, the responsible TLS polymerase frequently elicits untargeted mutagenesis as potentially a source of genetic diversity. Identifying such untargeted mutations in vivo is challenging due to the bulk of DNA that does not undergo TLS. Here, we present a protocol to enrich a plasmid pool that underwent Pol V-mediated TLS in Escherichia coli for mass sequencing. The concept of this protocol could be applied into any species. For complete details on the use and execution of this protocol, please refer to Isogawa et al. (2018).

Chromatin Pull-Down Methodology Based on DNA Triple Helix Formation.

Identification of the protein complexes associated with defined DNA sequence elements is essential to understand the numerous transactions in which DNA is involved, such as replication, repair, transcription, and chromatin dynamics. Here we describe two protocols, IDAP (Isolation of DNA Associated Proteins) and CoIFI (Chromatin-of-Interest Fragment Isolation), that allow for isolating DNA/protein complexes (i.e., nucleoprotein elements) by means of a DNA capture tool based on DNA triple helix (triplex) formation. Typically, IDAP is used to capture proteins that bind to a given DNA element of interest (e.g., a specific DNA sequence, an unusual DNA structure, a DNA lesion) that can be introduced at will into plasmids. The plasmids are immobilized by means of a triplex-forming probe on magnetic beads and incubated in nuclear extracts; by using in parallel a control plasmid (that lacks the DNA element of interest), proteins that preferentially bind to the DNA element of interest are captured and identified by mass spectrometry. Similarly, CoIFI also uses a triplex-forming probe to capture a specific chromatin fragment from a cultured cell line that has been engineered to contain multiple copies of the DNA element of interest.

Chronological Switch from Translesion Synthesis to Homology-Dependent Gap Repair In Vivo.

Cells are constantly exposed to endogenous and exogenous chemical and physical agents that damage their genome by forming DNA lesions. These lesions interfere with the normal functions of DNA such as transcription and replication, and need to be either repaired or tolerated. DNA lesions are accurately removed via various repair pathways. In contrast, tolerance mechanisms do not remove lesions but only allow replication to proceed despite the presence of unrepaired lesions. Cells possess two major tolerance strategies, namely translesion synthesis (TLS), which is an error-prone strategy and an accurate strategy based on homologous recombination (homology-dependent gap repair [HDGR]). Thus, the mutation frequency reflects the relative extent to which the two tolerance pathways operate in vivo. In the present paper, we review the present understanding of the mechanisms of TLS and HDGR and propose a novel and comprehensive view of the way both strategies interact and are regulated in vivo.

Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Post-replicative Gap Repair.

In vivo, replication forks proceed beyond replication-blocking lesions by way of downstream repriming, generating daughter strand gaps that are subsequently processed by post-replicative repair pathways such as homologous recombination and translesion synthesis (TLS). The way these gaps are filled during TLS is presently unknown. The structure of gap repair synthesis was assessed by sequencing large collections of single DNA molecules that underwent specific TLS events in vivo. The higher error frequency of specialized relative to replicative polymerases allowed us to visualize gap-filling events at high resolution. Unexpectedly, the data reveal that a specialized polymerase, Pol V, synthesizes stretches of DNA both upstream and downstream of a site-specific DNA lesion. Pol V-mediated untargeted mutations are thus spread over several hundred nucleotides, strongly eliciting genetic instability on either side of a given lesion. Consequently, post-replicative gap repair may be a source of untargeted mutations critical for gene diversification in adaptation and evolution.

Versatile and efficient chromatin pull-down methodology based on DNA triple helix formation.

The goal of present paper is to develop a reliable DNA-based method for isolation of protein complexes bound to DNA (Isolation of DNA Associated Proteins: IDAP). We describe a robust and versatile procedure to pull-down chromatinized DNA sequences-of-interest by formation of a triple helix between a sequence tag present in the DNA and a complementary triple helix forming oligonucleotide (TFO) coupled to a desthiobiotin residue. Following optimization to insure efficient recovery of native plasmids via TFO probe in vitro, the procedure is shown to work under various experimental situations. For instance, it allows capture proteins associated to plasmids hosted in E. coli, and is also successfully applied to recovering nucleosomes in vitro opening many possibilities to study post translational modifications of histones in a genuine nucleosome context. Incubation in human nuclear extracts of a plasmid carrying a NF-κB model promoter is shown to pull-down a specific transcription factor. Finally, isolation of a specific locus from human genomic chromatin has been successfully achieved (Chromatin-of-Interest Fragment Isolation: CoIFI). In conclusion, the methodology can be implemented for capturing proteins that specifically bind to any sequence-of-interest, DNA adduct or secondary structure provided a short sequence tag for triple helix formation is located nearby.

Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli.

It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM), exhibits several characteristics distinct from mutations that occur within the course of replication: i) following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min) than replication dependent mutations (t ½ ≈ 80-100 min) ii) NERiM specifically requires DNA Pol IV in addition to Pol V iii) NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure) of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication.

Roles of RecA protein in spontaneous mutagenesis in Escherichia coli.

To verify the extent of contribution of spontaneous DNA lesions to spontaneous mutagenesis, we have developed a new genetic system to examine simultaneously both forward mutations and recombination events occurring within about 600 base pairs of a transgenic rpsL target sequence located on Escherichia coli chromosome. In a wild-type strain, the recombination events were occurring at a frequency comparable to that of point mutations within the rpsL sequence. When the cells were UV-irradiated, the recombination events were induced much more sharply than point mutations. In a recA null mutant, no recombination event was observed. These data suggest that the blockage of DNA replication, probably caused by spontaneous DNA lesions, occurs often in normally growing E. coli cells and is mainly processed by cellular functions requiring the RecA protein. However, the recA mutant strain showed elevated frequencies of single-base frameshifts and large deletions, implying a novel mutator action of this strain. A similar mutator action of the recA mutant was also observed with a plasmid-based rpsL mutation assay. Therefore, if the recombinogenic problems in DNA replication are not properly processed by the RecA function, these would be a potential source for mutagenesis leading to single-base frameshift and large deletion in E. coli. Furthermore, the single-base frameshifts induced in the recA-deficient cells appeared to be efficiently suppressed by the mutS-dependent mismatch repair system. Thus, it seems likely that the single-base frameshifts are derived from slippage errors that are not directly caused by DNA lesions but made indirectly during some kind of error-prone DNA synthesis in the recA mutant cells.

RecFOR proteins are essential for Pol V-mediated translesion synthesis and mutagenesis.

When the replication fork moves through the template DNA containing lesions, daughter-strand gaps are formed opposite lesion sites. These gaps are subsequently filled-in either by translesion synthesis (TLS) or by homologous recombination. RecA filaments formed within these gaps are key intermediates for both of the gap-filling pathways. For instance, Pol V, the major lesion bypass polymerase in Escherichia coli, requires a functional interaction with the tip of the RecA filament. Here, we show that all three recombination mediator proteins RecFOR are needed to build a functionally competent RecA filament that supports efficient Pol V-mediated TLS in the presence of ssDNA-binding protein (SSB). A positive contribution of RecF protein to Pol V lesion bypass is demonstrated. When Pol III and Pol V are both present, Pol III imparts a negative effect on Pol V-mediated lesion bypass that is counteracted by the combined action of RecFOR and SSB. Mutations in recF, recO or recR gene abolish induced mutagenesis in E. coli.

Functional cooperation of Ogg1 and Mutyh in preventing G: C-->T: a transversions in mice.

Oxygen radicals generated through normal cellular metabolism induce a variety of types of oxidative damage into DNA and its precursors. Among such types of oxidative damage, 7, 8-dihydro-8-oxoguanine (8-oxoG), an oxidized form of guanine, is known to be abundant and highly mutagenic. 8-OxoG can pair with adenine as well as cytosine, thus causing G: C to T: A transversions after DNA replication, if not repaired. Organisms are equipped with elaborate systems to avoid such mutations caused by 8-oxoG. In Escherichia coli, two DNA glycosylases have been identified to suppress these mutations. One is MutM, an 8-oxoguanine DNA glycosylase that removes 8-oxoG from 8-oxoG: C base pairs. The other is MutY, an adenine DNA glycosylase that excises adenine from 8-oxoG: A mismatches. Mammals also have such DNA glycosylases; OGG1 (Ogg1) is the functional counterpart of MutM, and MUTYH (Mutyh) is the MutY homologue. In order to investigate the roles of these two enzymes in the avoidance of 8-oxoG-related mutagenesis in mammals, we analyzed spontaneous mutagenesis in the small intestine of Ogg1-deficient (Ogg1-/-) and Ogg1-, Mutyh-double deficient (Ogg1-/-; Mutyh-/-) mice at the age of 4-5 weeks using the prokaryotic rpsL transgene as a reporter. The observed mutation frequency was 1.00 x 10(-5) in both wild type and Ogg1-/- mice, and 1.91 x 10(-5) in Ogg1-/-; Mutyh-/- mice, indicating that the overall spontaneous mutation frequency was increased in Ogg1-/-; Mutyh-/- mice, but not in Ogg1-/- mice. Analysis of the mutation spectrum revealed that the frequency of G: C to T: A transversions were significantly increased in both Ogg1-/- and Ogg1-/-; Mutyh-/- mice; a 5-fold increase in Ogg1-/- mice, and a 41-fold increase in Ogg1-/-; Mutyh-/- mice when compared with wild type mice. A previous study in our laboratory indicated that a defect in Mutyh caused a 4-fold increase in the frequency of G: C to T: A transversions in mice. Combined, these observations suggest that a cooperative function between Ogg1 and Mutyh exists to prevent 8-oxoG-related mutagenesis in mammals.