Accidental Encounter of Repair Intermediates in Alkylated DNA May Lead to Double-Strand Breaks in Resting Cells.

In clinics, chemotherapy is often combined with surgery and radiation to increase the chances of curing cancers. In the case of glioblastoma (GBM), patients are treated with a combination of radiotherapy and TMZ over several weeks. Despite its common use, the mechanism of action of the alkylating agent TMZ has not been well understood when it comes to its cytotoxic effects in tumor cells that are mostly non-dividing.

Double-strand breaks: When DNA repair events accidentally meet.

The cellular response to alkylation damage is complex, involving multiple DNA repair pathways and checkpoint proteins, depending on the DNA lesion, the cell type, and the cellular proliferation state. The repair of and response to O-alkylation damage, primarily O-methylguaine DNA adducts (O-mG), is the purview of O-methylguanine-DNA methyltransferase (MGMT). Alternatively, this lesion, if left un-repaired, induces replication-dependent formation of the O-mG:T mis-pair and recognition of this mis-pair by the post-replication mismatch DNA repair pathway (MMR).

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 O-methylguanine (OmG) adducts. Current models propose that during DNA replication, thymine is incorporated across from OmG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double-strand breaks (DSBs).

Enrichment of plasmid pool from 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 is challenging due to the bulk of DNA that does not undergo TLS.

A Comprehensive View of Translesion Synthesis in Escherichia coli.

The lesion bypass pathway, translesion synthesis (TLS), exists in essentially all organisms and is considered a pathway for postreplicative gap repair and, at the same time, for lesion tolerance. As with the saying "a trip is not over until you get back home," studying TLS only at the site of the lesion is not enough to understand the whole process of TLS. Recently, a genetic study uncovered that polymerase V (Pol V), a poorly expressed TLS polymerase, is not only involved in the TLS step but also participates in the gap-filling reaction over several hundred nucleotides.

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.

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes.

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner.

PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching.

Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro.

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

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.

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.

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.

Inserting Site-Specific DNA Lesions into Whole Genomes.

Here, we describe a methodology that allows the insertion of site-specific DNA lesions into genomes in living cells. The technique involves the integration of a plasmid containing a site-specific lesion engineered in vitro into a precise location in the genome via the site-specific recombination reaction from phage lambda. The notion of DNA lesion is not restricted to chemically modified nucleotides but also refers to unusual DNA structures.

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.

Tolerance of lesions in E. coli: Chronological competition between Translesion Synthesis and Damage Avoidance.

Lesion tolerance pathways allow cells to proceed with replication despite the presence of replication-blocking lesions in their genome. Following transient fork stalling, replication resumes downstream leaving daughter strand gaps opposite replication-blocking lesions. The existence and repair of these gaps have been know for decades and are commonly referred to as postreplicative repair [39,38] (Rupp, 2013; Rupp and Howard-Flanders, 1968).

A defect in homologous recombination leads to increased translesion synthesis in E. coli.

DNA damage tolerance pathways allow cells to duplicate their genomes despite the presence of replication blocking lesions. Cells possess two major tolerance strategies, namely translesion synthesis (TLS) and homology directed gap repair (HDGR). TLS pathways involve specialized DNA polymerases that are able to synthesize past DNA lesions with an intrinsic risk of causing point mutations.

ExoMeg1: a new exonuclease from metagenomic library.

DNA repair mechanisms are responsible for maintaining the integrity of DNA and are essential to life. However, our knowledge of DNA repair mechanisms is based on model organisms such as Escherichia coli, and little is known about free living and uncultured microorganisms. In this study, a functional screening was applied in a metagenomic library with the goal of discovering new genes involved in the maintenance of genomic integrity.

Bacterial Proliferation: Keep Dividing and Don't Mind the Gap.

DNA Damage Tolerance (DDT) mechanisms help dealing with unrepaired DNA lesions that block replication and challenge genome integrity. Previous in vitro studies showed that the bacterial replicase is able to re-prime downstream of a DNA lesion, leaving behind a single-stranded DNA gap. The question remains of what happens to this gap in vivo.

The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity.

In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O(6)-methylguanine (O(6)MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O(6)-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown.

FF483-484 motif of human Polη mediates its interaction with the POLD2 subunit of Polδ and contributes to DNA damage tolerance.

Switching between replicative and translesion synthesis (TLS) DNA polymerases are crucial events for the completion of genomic DNA synthesis when the replication machinery encounters lesions in the DNA template. In eukaryotes, the translesional DNA polymerase η (Polη) plays a central role for accurate bypass of cyclobutane pyrimidine dimers, the predominant DNA lesions induced by ultraviolet irradiation. Polη deficiency is responsible for a variant form of the Xeroderma pigmentosum (XPV) syndrome, characterized by a predisposition to skin cancer.

Ethylene oxide and propylene oxide derived N7-alkylguanine adducts are bypassed accurately in vivo.

Adducts formed at the nucleophilic N7 position of guanine are the most abundant lesions produced by alkylating agents such as ethylene oxide (EO) and propylene oxide (PO). In order to investigate the intrinsic mutagenic potential of N7-alkylguanine adducts, we prepared single-stranded DNA probes containing a single well-defined N7-alkylguanine adduct under conditions that minimize the presence of depurinated molecules. Following introduction of these probes into Escherichia coli cells, the effect of the N7-alkylguanine adducts on the efficiency and fidelity of replication was determined.

Chronology in lesion tolerance gives priority to genetic variability.

The encounter of a replication fork with a blocking DNA lesion is a common event that cells need to address properly to preserve genome integrity. Cells possess two main strategies to tolerate unrepaired lesions: potentially mutagenic translesion synthesis (TLS) and nonmutagenic damage avoidance (DA). Little is known about the partitioning between these two strategies.

Translesion DNA synthesis and mutagenesis in prokaryotes.

The presence of unrepaired lesions in DNA represents a challenge for replication. Most, but not all, DNA lesions block the replicative DNA polymerases. The conceptually simplest procedure to bypass lesions during DNA replication is translesion synthesis (TLS), whereby the replicative polymerase is transiently replaced by a specialized DNA polymerase that synthesizes a short patch of DNA across the site of damage.

Escherichia coli DNA polymerase III is responsible for the high level of spontaneous mutations in mutT strains.

Reactive oxygen species induce oxidative damage in DNA precursors, i.e. dNTPs, leading to point mutations upon incorporation.

Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome.

Although most deoxyribonucleic acid (DNA) lesions are accurately repaired before replication, replication across unrepaired lesions is the main source of point mutations. The lesion tolerance processes, which allow damaged DNA to be replicated, entail two branches, error-prone translesion synthesis (TLS) and error-free damage avoidance (DA). While TLS pathways are reasonably well established, DA pathways are poorly understood.