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). This paper analyzes the interaction of the pathways involved in the repair of these gaps. A key repair intermediated is formed when RecA protein binds to these gaps forming ssDNA.RecA filaments establishing the so-called SOS signal. The gaps are either “repaired” by Translesion Synthesis (TLS), a process that involves the transient recruitment of a specialized DNA polymerase that copies the lesion with an intrinsic risk of fixing a mutation opposite the lesion site, or by Damage Avoidance, an error-free pathway that involves homologous recombination with the sister chromatid (Homology Directed Gap Repair: HDGR). We have developed an assay that allows one to study the partition between TLS and HDGR in the context of a single replication-blocking lesion present in the E. coli chromosome. The level of expression of the TLS polymerases controls the extent of TLS. Our data show that TLS is implemented first with great parsimony, followed by abundant recombination-based tolerance events. Indeed, the substrate for TLS, i.e., the ssDNA.RecA filament, persists for only a limited amount of time before it engages in an early recombination intermediates (D-loop) with the sister chromatid. Time-based competition between TLS and HDGR is set by mere sequestration of the TLS substrates into early recombination intermediates. Most gaps are subsequently repaired by Homology Directed Gap Repair (HDGR), a pathway that involves RecA. Surprisingly, however, in the absence of RecA, some cells manage to divide and form colonies at the expense of losing the damage-containing chromatid.
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. Following the insertion of a single lesion in the chromosome of a living cell, we showed that this gap is mostly filled in by Homology Directed Gap Repair in a RecA dependent manner. When cells fail to repair this gap, or when homologous recombination is impaired, cells are still able to divide, leading to the loss of the damaged chromatid, suggesting that bacteria lack a stringent cell division checkpoint mechanism. Hence, at the expense of losing one chromatid, cell survival and proliferation are ensured.
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. The same study revealed that in contrast, Pol IV, another highly expressed TLS polymerase, essentially stays away from the gap-filling reaction. These observations imply fundamentally different ways these polymerases are recruited to DNA in cells. While access of Pol IV appears to be governed by mass action, efficient recruitment of Pol V involves a chaperone-like action of the RecA filament. We present a model of Pol V activation: the 3' tip of the RecA filament initially stabilizes Pol V to allow stable complex formation with a sliding β-clamp, followed by the capture of the terminal RecA monomer by Pol V, thus forming a functional Pol V complex. This activation process likely determines higher accessibility of Pol V than of Pol IV to normal DNA. Finally, we discuss the biological significance of TLS polymerases during gap-filling reactions: error-prone gap-filling synthesis may contribute as a driving force for genetic diversity, adaptive mutation, and evolution.
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. This process is inherently error prone and is the main source of point mutations. The diversity of existing DNA lesions and the biochemical properties of Escherichia coli DNA polymerases will be presented. Our main goal is to deliver an integrated view of TLS pathways involving the multiple switches between replicative and specialized DNA polymerases and their interaction with key accessory factors. Finally, a brief glance at how other bacteria deal with TLS and mutagenesis is presented.
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 O6-methylguaine DNA adducts (O6-mG), is the purview of O6-methylguanine-DNA methyltransferase (MGMT). Alternatively, this lesion, if left un-repaired, induces replication-dependent formation of the O6-mG:T mis-pair and recognition of this mis-pair by the post-replication mismatch DNA repair pathway (MMR). Two models have been suggested to account for MMR and O6-mG DNA lesion dependent formation of DNA double-strand breaks (DSBs) and the resulting cytotoxicity – futile cycling and direct DNA damage signaling. While there have been hints at crosstalk between the MMR and base excision repair (BER) pathways, clear mechanistic evidence for such pathway coordination in the formation of DSBs has remained elusive. However, using a novel protein capture approach, Fuchs and colleagues have demonstrated that DSBs result from an encounter between MMR-induced gaps initiated at alkylation induced O6-mG:C sites and BER-induced nicks at nearby N-alkylation adducts in the opposite strand. The accidental encounter between these two repair events is causal in the formation of DSBs and the resulting cellular response, documenting a third model to account for O6-mG induced cell death in non-replicating cells. This graphical review highlights the details of this Repair Accident model, as compared to current models, and we discuss potential strategies to improve clinical use of alkylating agents such as temozolomide, that can be inferred from the Repair Accident model.
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. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38-/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38-/- cells did not show a significant sensitivity to either UV or H2O2, a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38-/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38-/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.
The replicative bypass of base damage in DNA (translesion DNA synthesis TLS) is a ubiquitous mechanism for relieving arrested DNA replication. The process requires multiple polymerase switching ...events during which the high-fidelity DNA polymerase in the replication machinery arrested at the primer terminus is replaced by one or more polymerases that are specialized for TLS. When replicative bypass is fully completed, the primer terminus is once again occupied by high-fidelity polymerases in the replicative machinery. This review addresses recent advances in our understanding of DNA polymerase switching during TLS in bacteria such as
E. coli and in lower and higher eukaryotes.
Genomes of all living organisms are constantly injured by endogenous and exogenous agents that modify the chemical integrity of DNA and in turn challenge its informational content. Despite the ...efficient action of numerous repair systems that remove lesions in DNA in an error-free manner, some lesions, that escape these repair mechanisms, are present when DNA is being replicated. Although replicative DNA polymerases are usually unable to copy past such lesions, it was recently discovered that cells are equipped with specialized DNA polymerases that will assist the replicative polymerase during the process of Translesion Synthesis (TLS). These TLS polymerases exhibit relaxed fidelity that allows them to copy past lesions in DNA with an inherent risk of generating mutations at high frequency. We present recent aspects related to the genetics and biochemistry of TLS and highlight some of the remaining hot topics of this field.
Most current models for replication past damaged lesions envisage that translesion synthesis occurs at the replication fork. However older models suggested that gaps were left opposite lesions to ...allow the replication fork to proceed, and these gaps were subsequently sealed behind the replication fork. Two recent articles lend support to the idea that bypass of the damage occurs behind the fork. In the first paper, electron micrographs of DNA replicated in UV-irradiated yeast cells show regions of single-stranded DNA both at the replication forks and behind the fork, the latter being consistent with the presence of gaps in the daughter-strands opposite lesions. The second paper describes an
in vitro DNA replication system reconstituted from purified bacterial proteins. Repriming of synthesis downstream from a blocked fork occurred not only on the lagging strand as expected, but also on the leading strand, demonstrating that contrary to widely accepted beliefs, leading strand synthesis does not need to be continuous.
Numerous agents attack DNA, forming lesions that impair normal replication. Specialized DNA polymerases transiently replace the replicative polymerase and copy past lesions, thus generating ...mutations, the major initiating cause of cancer. We monitored, in Escherichia coli, the kinetics of replication of both strands of DNA molecules containing a single replication block in either the leading or lagging strand. Despite a block in the leading strand, lagging-strand synthesis proceeded further, implying transient uncoupling of concurrent strand synthesis. Replication through the lesion requires specialized DNA polymerases and is achieved with similar kinetics and efficiencies in both strands.
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