Maintenance Of Genome Integrity Research Articles

Imbalance in the antioxidant defence system and pro-genotoxic status induced by high glucose concentrations: In vitro testing in human liver cells

It has been hypothesized that high glucose concentrations might contribute to the overall intracellular oxidative stress either by the direct generation of reactive oxygen species (ROS) or by altering the redox balance. Moreover, it has also been suggested that high glucose concentration can increase the susceptibility of DNA to genotoxic effects of xenobiotics. The aim of this approach was to test high glucose concentrations for pro-genotoxicity in human liver cells by setting up an in vitro model for hyperglycaemia. The experimental design included performing of tests on both human HepG2 tumour cells and HepaRG immortalized cells. Increased cell susceptibility to genotoxic xenobiotics was tested by challenging cell cultures with 4-nitroquinoline-N-oxide (4NQO) and evaluating the extent of primary DNA damage by comet assay. Moreover, we evaluated the relationship between glucose concentration and intracellular ROS, as well as the effects of glucose concentration on the induction of Nrf2-dependent genes such as Glutathione S-transferases, Heme‑oxygenase-1, and Glutathione peroxidase-4. To investigate the involvement of ROS in the induced pro-genotoxic activity, parallel experimental sets were set up by considering co-treatment of cells with the model mutagen 4NQO and the antioxidant, glutathione precursor N-acetyl-L-cysteine. High glucose concentrations caused a significant increase in the levels of primary DNA damage, with a pro-genotoxic condition closely related to the concentration of glucose in the culture medium when cells were exposed to 4NQO. High glucose concentrations also stimulated the production of ROS and down-regulated genes involved in contrasting of the effects of oxidative stress. In conclusion, in the presence of high concentrations of glucose, the cells are in unfavourable conditions for the maintenance of genome integrity.

Measuring Genome-Wide Nascent Nucleosome Assembly Using ReIN-Map.

The successful assembly of nucleosomes following DNA replication is critically important for both the inheritance of epigenetic information and the maintenance of genome integrity. This process, termed DNA replication-coupled (RC) nucleosome assembly, requires that DNA replication and nucleosome assembly function in a highly coordinated fashion to transmit both genetic and epigenetic information. In this chapter, we describe a genome-wide method for measuring nucleosome occupancy patterns on nascent strands, which we have termed Replication-Intermediate Nucleosome Mapping (ReIN-Map), to monitor the RC nucleosome assembly level genome-wide in vivo. This method takes advantage of next-generation sequencing and in vivo labeling of newly synthesized DNA using a thymidine analogue, 5-bromo-2'-deoxyuridine (BrdU), and involves parallel analyses of the nucleosome formation using micrococcal nuclease (MNase) digestion of chromatin (MNase-seq) and of the newly synthesized DNA levels using sonication shearing of chromatin s (Sonication-seq). Replicated chromatin was enriched by immunoprecipitation using antibodies against BrdU (BrdU-IP), which is incorporated into DNA during DNA synthesis; the DNA is then subjected to strand-specific sequencing.

Genome maintenance functions of a putative Trypanosoma brucei translesion DNA polymerase include telomere association and a role in antigenic variation.

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.

Fluorescence Microscopy for Analysis of Relocalization of Structure-Specific Endonucleases.

The analysis of protein relocalization by fluorescence microscopy has been important for studying processes involved in genome integrity maintenance at the cellular level. Structure-specific endonucleases are required for genome stability, and work in budding yeast has revealed that these proteins accumulate and colocalize at discrete subnuclear foci following DNA damage. Here we describe protocols for fluorescence microscopy analysis of live budding-yeast cells containing fluorescent-tagged proteins that have been useful for the study of endonuclease relocalization during the cell cycle and under DNA-damaging conditions, all of which can be extended to the analysis of other proteins.

Autophagy Contributes to the Maintenance of Genomic Integrity by Reducing Oxidative Stress.

Autophagy has been well documented to play an important role in maintaining genomic stability. However, in addition to directly engulfing and digesting the damaged organelles and chromatin fragments, autophagy can affect many cellular processes including DNA damage response, regulation of redox homeostasis, and cell division; it remains to be determined to what extent each of those processes contributes to the maintenance of genomic stability. We here examined the role of autophagy-dependent redox regulation in the maintenance of genomic stability in two cancer cell lines (HT1080 and U2OS) and mesenchymal stem cells (MSCs) using micronuclei MN, also referred to as cytoplasmic chromatin fragments, as a marker. Our results showed that the spontaneous and genotoxic stress-induced frequencies of MN in cancer cells were significantly reduced by autophagy activators rapamycin and Torin1, and the reduction in MN was accompanied by a reduction in reactive oxygen species (ROS). Increased micronucleation in senescent MSCs, in which autophagic flux is blocked, was also attenuated by rapamycin, together with a reduction in ROS. Inhibition of autophagy by chloroquine (CQ) or ATG5 depletion, on the other hand, resulted in an increased frequency of MN, though a ROS elevation in response to autophagy inhibition was only observed in MSCs. Importantly, the induction of MN by autophagy inhibition in MSCs could be abrogated by antioxidant N-acetylcysteine (NAC). In contrast to the reported impairment of CHK1 activation in Atg7-deficient mouse embryonic fibroblasts, we found that the level of phosphorylated CHK1 was increased by CQ or ATG5 depletion but decreased by rapamycin or Torin1, suggesting that the increased genomic instability by defective autophagy is not caused by insufficient activation of CHK1-homologous recombination cascade. Together, our findings suggest that redox homeostasis regulated by autophagy contributes substantially to the maintenance of genomic stability in certain contexts.

Temporal activation of LRH-1 and RAR-γ in human pluripotent stem cells induces a functional naïve-like state.

Naïve pluripotency can be established in human pluripotent stem cells (hPSCs) by manipulation of transcription factors, signaling pathways, or a combination thereof. However, differences exist in the molecular and functional properties of naïve hPSCs generated by different protocols, which include varying similarities with pre-implantation human embryos, differentiation potential, and maintenance of genomic integrity. We show here that short treatment with two chemical agonists (2a) of nuclear receptors, liver receptor homologue-1 (LRH-1) and retinoic acid receptor gamma (RAR-γ), along with 2i/LIF (2a2iL) induces naïve-like pluripotency in human cells during reprogramming of fibroblasts, conversion of pre-established hPSCs, and generation of new cell lines from blastocysts. 2a2iL-hPSCs match several defined criteria of naïve-like pluripotency and contribute to human-mouse interspecies chimeras. Activation of TGF-β signaling is instrumental for acquisition of naïve-like pluripotency by the 2a2iL induction procedure, and transient activation of TGF-β signaling substitutes for 2a to generate naïve-like hPSCs. We reason that 2a2iL-hPSCs are an easily attainable system to evaluate properties of naïve-like hPSCs and for various applications.

DNA-PKcs Ser2056 auto-phosphorylation is affected by an O-GlcNAcylation/phosphorylation interplay

BackgroundDNA dependent Protein Kinase (DNA-PK) is an heterotrimeric complex regulating the Non Homologous End Joining (NHEJ) double strand break (DSB) repair pathway. The activity of its catalytic subunit (DNA-PKcs) is regulated by multiple phosphorylations, like the Ser2056 one that impacts DSB end processing and telomeres integrity. O-GlcNAcylation is a post translational modification (PTM) closely related to phosphorylation and its implication in the modulation of DNA-PKcs activity during the DNA Damage Response (DDR) is unknown. MethodsUsing IP techniques, and HeLa cell line, we evaluated the effect of pharmacological or siOGT mediated O-GlcNAc level modulation on DNA-PKcs O-GlcNAcylation. We used the RPA32 phosphorylation as a DNA-PKcs activity reporter substrate to evaluate the effect of O-GlcNAc modulators. ResultsWe show here that human DNA-PKcs is an O-GlcNAc modified protein and that this new PTM is responsive to the cell O-GlcNAcylation level modulation. Our findings reveal that DNA-PKcs hypo O-GlcNAcylation affects its kinase activity and that the bleomycin-induced Ser2056 phosphorylation, is modulated by DNA-PKcs O-GlcNAcylation. ConclusionsDNA-PKcs Ser2056 phosphorylation is antagonistically linked to DNA-PKcs O-GlcNAcylation level modulation. General significanceGiven the essential role of DNA-PKcs Ser2056 phosphorylation in the DDR, this study brings data about the role of cell O-GlcNAc level on genome integrity maintenance.

Arabidopsis BRCA1 represses RRTF1-mediated ROS production and ROS-responsive gene expression under dehydration stress.

Reactive oxygen species (ROS) act as important secondary messengers in abscisic acid (ABA) signaling and induce stomatal closure under dehydration stress. The breast cancer susceptibility gene 1 (BRCA1), an important tumor suppressor in animals, functions primarily in the maintenance of genome integrity in animals and plants. However, whether and how the plant BRCA1 regulates intracellular ROS homeostasis in guard cells under dehydration stress remains unknown. Here, we found that Arabidopsisatbrca1 loss-of-function mutants showed dehydration stress tolerance. This stress tolerant phenotype of atbrca1 was a result of ABA- and ROS-induced stomatal closure, which was enhanced in atbrca1 mutants compared with the wild-type. AtBRCA1 downregulated the expression of ROS-responsive and marker genes. Notably, these genes were also the targets of the AP2/ERF transcriptional activator RRTF1/ERF109. Under normal conditions, AtBRCA1 physically interacted with RRTF1 and inhibited its binding to the GCC-box-like sequence in target gene promoters. Under dehydration stress, the expression of AtBRCA1 was dramatically reduced and that of RRTF1 was activated, thus inducing the expression of ROS-responsive genes. Overall, our study reveals a novel molecular function of AtBRCA1 in the transcriptional regulation of intracellular ROS homeostasis under dehydration stress.

Rapid progressive ALS in a patient with a DNAJC7 loss-of-function mutation.

ABSTRACT In S. cerevisiae, the Pif1 helicase functions to impact both nuclear and mitochondrial DNA replication and repair processes. Pif1 is a 5’-3’ helicase, which preferentially unwinds RNA-DNA hybrids and resolves G-quadruplex structures. Further, regulation of Pif1 by phosphorylation negatively impacts its interaction with telomerase during double strand break repair. Here, we report that in addition to phosphorylation, Pif1 is also modified by lysine acetylation, which influences both its cellular and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 (Esa1) and deacetylase Rpd3 are primarily responsible for dynamically acetylating nuclear Pif1. Mass spectrometry analysis revealed that Pif1 was modified throughout the protein’s sequence on the N-terminus (K118, K129), helicase domain (K525, K639, K725), and C-terminus (K800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N-terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase activity, while maintaining its substrate preferences. Additionally, both the ATPase and DNA binding activities of Pif1 were stimulated upon acetylation. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose an acetylation-based model for the regulation of Pif1 activities, addressing how this post translational modification can influence its role as a key player in a multitude of DNA transactions vital to the maintenance of genome integrity.

Mgs1 protein supports genome stability via recognition of G-quadruplex DNA structures.

The integrity of the genetic material is crucial for every organism. One intrinsic attack to genome stability is stalling of the replication fork which can result in DNA breakage. Several factors, such as DNA lesions or the formation of stable secondary structures (eg, G-quadruplexes) can lead to replication fork stalling. G-quadruplexes (G4s) are well-characterized stable secondary DNA structures that can form within specific single-stranded DNA sequence motifs and have been shown to block/pause the replication machinery. In most genomes several helicases have been described to regulate G4 unfolding to preserve genome integrity, however, different experiments raise the hypothesis that processing of G4s during DNA replication is more complex and requires additional, so far unknown, proteins. Here, we show that the Saccharomyces cerevisiae Mgs1 protein robustly binds to G4 structures in vitro and preferentially acts at regions with a strong potential to form G4 structures in vivo. Our results suggest that Mgs1 binds to G4-forming sites and has a role in the maintenance of genome integrity.

Interplay between Cellular Metabolism and the DNA Damage Response in Cancer.

Metabolism is a fundamental cellular process that can become harmful for cells by leading to DNA damage, for instance by an increase in oxidative stress or through the generation of toxic byproducts. To deal with such insults, cells have evolved sophisticated DNA damage response (DDR) pathways that allow for the maintenance of genome integrity. Recent years have seen remarkable progress in our understanding of the diverse DDR mechanisms, and, through such work, it has emerged that cellular metabolic regulation not only generates DNA damage but also impacts on DNA repair. Cancer cells show an alteration of the DDR coupled with modifications in cellular metabolism, further emphasizing links between these two fundamental processes. Taken together, these compelling findings indicate that metabolic enzymes and metabolites represent a key group of factors within the DDR. Here, we will compile the current knowledge on the dynamic interplay between metabolic factors and the DDR, with a specific focus on cancer. We will also discuss how recently developed high-throughput technologies allow for the identification of novel crosstalk between the DDR and metabolism, which is of crucial importance to better design efficient cancer treatments.

Investigating the pathogenic SNPs in BLM helicase and their biological consequences by computational approach

The BLM helicase protein plays a vital role in DNA replication and the maintenance of genomic integrity. Variation in the BLM helicase gene resulted in defects in the DNA repair mechanism and was reported to be associated with Bloom syndrome (BS) and cancer. Despite extensive investigation of helicase proteins in humans, no attempt has previously been made to comprehensively analyse the single nucleotide polymorphism (SNPs) of the BLM gene. In this study, a comprehensive analysis of SNPs on the BLM gene was performed to identify, characterize and validate the pathogenic SNPs using computational approaches. We obtained SNP data from the dbSNP database version 150 and mapped these data to the genomic coordinates of the “NM_000057.3” transcript expressing BLM helicase (P54132). There were 607 SNPs mapped to missense, 29 SNPs mapped to nonsense, and 19 SNPs mapped to 3′-UTR regions. Initially, we used many consensus tools of SIFT, PROVEAN, Condel, and PolyPhen-2, which together increased the accuracy of prediction and identified 18 highly pathogenic non-synonymous SNPs (nsSNPs) out of 607 SNPs. Subsequently, these 18 high-confidence pathogenic nsSNPs were analysed for BLM protein stability, structure–function relationships and disease associations using various bioinformatics tools. These 18 mutants of the BLM protein along with the native protein were further investigated using molecular dynamics simulations to examine the structural consequences of the mutations, which might reveal their malfunction and contribution to disease. In addition, 28 SNPs were predicted as “stop gained” nonsense SNPs and one SNP was predicted as “start lost”. Two SNPs in the 3′UTR were found to abolish miRNA binding and thus may enhance the expression of BLM. Interestingly, we found that BLM mRNA overexpression is associated with different types of cancers. Further investigation showed that the dysregulation of BLM is associated with poor overall survival (OS) for lung and gastric cancer patients and hence led to the conclusion that BLM has the potential to be used as an important prognostic marker for the detection of lung and gastric cancer.

The SIRT6 activator MDL-800 improves genomic stability and pluripotency of old murine-derived iPS cells.

Cellular reprogramming is an emerging strategy for delaying the aging processes. However, a number of challenges, including the impaired genome integrity and decreased pluripotency of induced pluripotent stem cells (iPSCs) derived from old donors, may hinder their potential clinical applications. The longevity gene, Sirtuin 6 (SIRT6), functions in multiple biological processes such as the maintenance of genome integrity and the regulation of somatic cell reprogramming. Here, for the first time, we demonstrate that MDL‐800, a recently developed selective SIRT6 activator, improved genomic stability by activating two DNA repair pathways—nonhomologous end joining (NHEJ) and base excision repair (BER) in old murine‐derived iPSCs. More interestingly, we found that pretreating old murine iPSCs, which normally exhibit a restricted differentiation potential, with MDL‐800 promoted the formation of teratomas comprised of all three germ layers and robustly stimulated chimera generation. Our findings suggest that pharmacological activation of SIRT6 holds great promise in treating aging‐associated diseases with iPSC‐based cell therapy.

Mthfd2 Modulates Mitochondrial Function and DNA Repair to Maintain the Pluripotency of Mouse Stem Cells.

SummaryThe pluripotency of stem cells determines their developmental potential. While the pluripotency states of pluripotent stem cells are variable and interconvertible, the mechanisms underlying the acquisition and maintenance of pluripotency remain largely elusive. Here, we identified that methylenetetrahydrofolate dehydrogenase (NAD+-dependent), methenyltetrahydrofolate cyclohydrolase (Mthfd2) plays an essential role in maintaining embryonic stem cell pluripotency and promoting complete reprogramming of induced pluripotent stem cells. Mechanistically, in mitochondria, Mthfd2 maintains the integrity of the mitochondrial respiratory chain and prevents mitochondrial dysfunction. In the nucleus, Mthfd2 stabilizes the phosphorylation of EXO1 to support DNA end resection and promote homologous recombination repair. Our results revealed that Mthfd2 is a dual-function factor in determining the pluripotency of pluripotent stem cells through both mitochondrial and nuclear pathways, ultimately ensuring safe application of pluripotent stem cells.

Guarding the Genome: CENP-A-Chromatin in Health and Cancer.

Faithful chromosome segregation is essential for the maintenance of genomic integrity and requires functional centromeres. Centromeres are epigenetically defined by the histone H3 variant, centromere protein A (CENP-A). Here we highlight current knowledge regarding CENP-A-containing chromatin structure, specification of centromere identity, regulation of CENP-A deposition and possible contribution to cancer formation and/or progression. CENP-A overexpression is common among many cancers and predicts poor prognosis. Overexpression of CENP-A increases rates of CENP-A deposition ectopically at sites of high histone turnover, occluding CCCTC-binding factor (CTCF) binding. Ectopic CENP-A deposition leads to mitotic defects, centromere dysfunction and chromosomal instability (CIN), a hallmark of cancer. CENP-A overexpression is often accompanied by overexpression of its chaperone Holliday Junction Recognition Protein (HJURP), leading to epigenetic addiction in which increased levels of HJURP and CENP-A become necessary to support rapidly dividing p53 deficient cancer cells. Alterations in CENP-A posttranslational modifications are also linked to chromosome segregation errors and CIN. Collectively, CENP-A is pivotal to genomic stability through centromere maintenance, perturbation of which can lead to tumorigenesis.

Telomere shortening in patients with drug-resistant epilepsy

Epilepsy affects about 1 % of the world population. Mesial temporal lobe epilepsy (mTLE) presents with seizures initiated in hippocampus and is the most frequent form of epilepsy. About 30 % of individuals with mTLE do not respond to conventional medications maintaining seizures and consequently new lesions on a daily basis. Treatment-resistant epilepsy has a huge social and individual burden due to impaired quality of life and increased mortality rate.There are many reasons for telomere shortening in individuals with mTLE, such as a chronic mitochondrial oxidative stress and increased levels of pro-inflammatory mediators. In the past 10 years, there was a boom of studies establishing association between telomere length and chronic/complex disorders. Telomeres are essential for the maintenance of genomic integrity. Telomere length has been assumed as a biological marker for stress and cellular ageing. Here we hypothesized that individuals affected with treatment-resistant mTLE would course with a shorter telomere than controls. So, we measured leucocytes telomere length in a sample of 89 individuals, 48 treatment-resistant mTLE compared to 41 healthy controls. As expected, we observed a significant shorter telomere in the peripheral cell leukocytes of treatment-resitant mTLE group. Telomere length was not associated with sex, side of hippocampal sclerosis, family history, etiology of seizures, duration of disease or the Engel score. Our results points towards the need of further investigation to shed light on the relation of telomeres shortening and the outcomes and impacts of epilepsy.

Jack of all trades? The versatility of RNA in DNA double-strand break repair.

The mechanisms by which RNA acts in the DNA damage response (DDR), specifically in the repair of DNA double-strand breaks (DSBs), are emerging as multifaceted and complex. Different RNA species, including but not limited to; microRNA (miRNA), long non-coding RNA (lncRNA), RNA:DNA hybrid structures, the recently identified damage-induced lncRNA (dilncRNA), damage-responsive transcripts (DARTs), and DNA damage-dependent small RNAs (DDRNAs), have been shown to play integral roles in the DSB response. The diverse properties of these RNAs, such as sequence, structure, and binding partners, enable them to fulfil a variety of functions in different cellular contexts. Additionally, RNA can be modified post-transcriptionally, a process which is regulated in response to cellular stressors such as DNA damage. Many of these mechanisms are not yet understood and the literature contradictory, reflecting the complexity and expansive nature of the roles of RNA in the DDR. However, it is clear that RNA is pivotal in ensuring the maintenance of genome integrity. In this review, we will discuss and summarise recent evidence which highlights the roles of these various RNAs in preserving genomic integrity, with a particular focus on the emerging role of RNA in the DSB repair response.

SUMOylation stabilizes hSSB1 and enhances the recruitment of NBS1 to DNA damage sites

Human single-stranded DNA-binding protein 1 (hSSB1) is required for the efficient recruitment of the MRN complex to DNA double-strand breaks and is essential for the maintenance of genome integrity. However, the mechanism by which hSSB1 recruits NBS1 remains elusive. Here, we determined that hSSB1 undergoes SUMOylation at both K79 and K94 under normal conditions and that this modification is dramatically enhanced in response to DNA damage. SUMOylation of hSSB1, which is specifically fine-tuned by PIAS2α, and SENP2, not only stabilizes the protein but also enhances the recruitment of NBS1 to DNA damage sites. Cells with defective hSSB1 SUMOylation are sensitive to ionizing radiation, and global inhibition of SUMOylation by either knocking out UBC9 or adding SUMOylation inhibitors significantly enhances the sensitivity of cancer cells to etoposide. Our findings reveal that SUMOylation, as a novel posttranslational modification of hSSB1, is critical for the functions of this protein, indicating that the use of SUMOylation inhibitors (e.g., 2-D08 and ML-792) may be a new strategy that would benefit cancer patients being treated with chemo- or radiotherapy.

Human RTEL1 associates with Poldip3 to facilitate responses to replication stress and R-loop resolution.

RTEL1 helicase is a component of DNA repair and telomere maintenance machineries. While RTEL1's role in DNA replication is emerging, how RTEL1 preserves genomic stability during replication remains elusive. Here we used a range of proteomic, biochemical, cell, and molecular biology and gene editing approaches to provide further insights into potential role(s) of RTEL1 in DNA replication and genome integrity maintenance. Our results from complementary human cell culture models established that RTEL1 and the Polδ subunit Poldip3 form a complex and are/function mutually dependent in chromatin binding after replication stress. Loss of RTEL1 and Poldip3 leads to marked R-loop accumulation that is confined to sites of active replication, enhances endogenous replication stress, and fuels ensuing genomic instability. The impact of depleting RTEL1 and Poldip3 is epistatic, consistent with our proposed concept of these two proteins operating in a shared pathway involved in DNA replication control under stress conditions. Overall, our data highlight a previously unsuspected role of RTEL1 and Poldip3 in R-loop suppression at genomic regions where transcription and replication intersect, with implications for human diseases including cancer.

Visualizing mutagenic repair: novel insights into bacterial translesion synthesis.

ABSTRACTDNA repair is essential for cell survival. In all domains of life, error-prone and error-free repair pathways ensure maintenance of genome integrity under stress. Mutagenic, low-fidelity repair mechanisms help avoid potential lethality associated with unrepaired damage, thus making them important for genome maintenance and, in some cases, the preferred mode of repair. However, cells carefully regulate pathway choice to restrict activity of these pathways to only certain conditions. One such repair mechanism is translesion synthesis (TLS), where a low-fidelity DNA polymerase is employed to synthesize across a lesion. In bacteria, TLS is a potent source of stress-induced mutagenesis, with potential implications in cellular adaptation as well as antibiotic resistance. Extensive genetic and biochemical studies, predominantly in Escherichia coli, have established a central role for TLS in bypassing bulky DNA lesions associated with ongoing replication, either at or behind the replication fork. More recently, imaging-based approaches have been applied to understand the molecular mechanisms of TLS and how its function is regulated. Together, these studies have highlighted replication-independent roles for TLS as well. In this review, we discuss the current status of research on bacterial TLS, with emphasis on recent insights gained mostly through microscopy at the single-cell and single-molecule level.