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Translesion DNA polymerases are required for spontaneous deletion formation in Salmonella typhimurium.

ABSTRACT: How spontaneous deletions form in bacteria is still a partly unresolved problem. Here, we show that deletion formation in Salmonella typhimurium requires the presence of functional translesion polymerases. First, in wild-type bacteria, removal of the known translesion DNA polymerases, PolII (polB), PolIV (dinB), PolV (umuDC), and SamAB (samAB), resulted in a 10-fold decrease in the deletion rate, indicating that 90% of all spontaneous deletions require these polymerases for their formation. Second, overexpression of these polymerases by derepression of the DNA damage-inducible LexA regulon caused a 25-fold increase in deletion rate that depended on the presence of functional translesion polymerases. Third, overexpression of the polymerases PolII and PolIV from a plasmid increased the deletion rate 12- to 30-fold, respectively. Last, in a recBC(-) mutant where dsDNA ends are stabilized due to the lack of the end-processing nuclease RecBC, the deletion rate was increased 20-fold. This increase depended on the translesion polymerases. In lexA(def) mutant cells with constitutive SOS expression, a 10-fold increase in DNA breaks was observed. Inactivation of all 4 translesion polymerases in the lexA(def) mutant reduced the deletion rate 250-fold without any concomitant reduction in the amount of DNA breaks. Mutational inactivation of 3 endonucleases under LexA control reduced the number of DNA breaks to the wild-type level in a lexA(def) mutant with a concomitant 50-fold reduction in deletion rate. These findings suggest that the translesion polymerases are not involved in forming the DNA breaks, but that they require them to stimulate deletion formation.

SUBMITTER: Koskiniemi S

PROVIDER: S-EPMC2700912 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Translesion DNA polymerases.

Project description:Living cells are continually exposed to DNA-damaging agents that threaten their genomic integrity. Although DNA repair processes rapidly target the damaged DNA for repair, some lesions nevertheless persist and block genome duplication by the cell's replicase. To avoid the deleterious consequence of a stalled replication fork, cells use specialized polymerases to traverse the damage. This process, termed "translesion DNA synthesis" (TLS), affords the cell additional time to repair the damage before the replicase returns to complete genome duplication. In many cases, this damage-tolerance mechanism is error-prone, and cell survival is often associated with an increased risk of mutagenesis and carcinogenesis. Despite being tightly regulated by a variety of transcriptional and posttranslational controls, the low-fidelity TLS polymerases also gain access to undamaged DNA where their inaccurate synthesis may actually be beneficial for genetic diversity and evolutionary fitness.

| S-EPMC3783050 | biostudies-literature

Translesion DNA polymerases and cancer.

Project description:DNA repair has been regarded as an important barrier to carcinogenesis. The newly discovered field of translesion synthesis (TLS) has made it apparent that mammalian cells need distinct polymerases to efficiently and accurately bypass DNA lesions. Perturbation of TLS polymerase activity by mutation, loss of expression, etc. is expected to result in the accumulation of mutations in cells exposed to specific carcinogens. Furthermore, several TLS polymerases can modulate cellular sensitivity to chemotherapeutic agents. TLS genes and TLS gene variations may thus be attractive pharmacologic and/or pharmacogenetic targets. We review herein current data with regards to the potential contribution of the primary TLS polymerase genes to cancer, their interaction with pharmacologic agents, and identify areas of interest for further research.

| S-EPMC3434439 | biostudies-literature

Plant organellar DNA polymerases are replicative and translesion DNA synthesis polymerases.

Project description:Genomes acquire lesions that can block the replication fork and some lesions must be bypassed to allow survival. The nuclear genome of flowering plants encodes two family-A DNA polymerases (DNAPs), the result of a duplication event, that are the sole DNAPs in plant organelles. These DNAPs, dubbed Plant Organellar Polymerases (POPs), resemble the Klenow fragment of bacterial DNAP I and are not related to metazoan and fungal mitochondrial DNAPs. Herein we report that replicative POPs from the plant model Arabidopsis thaliana (AtPolI) efficiently bypass one the most insidious DNA lesions, an apurinic/apyrimidinic (AP) site. AtPolIs accomplish lesion bypass with high catalytic efficiency during nucleotide insertion and extension. Lesion bypass depends on two unique polymerization domain insertions evolutionarily unrelated to the insertions responsible for lesion bypass by DNAP ?, an analogous lesion bypass polymerase. AtPolIs exhibit an insertion fidelity that ranks between the fidelity of replicative and lesion bypass DNAPs, moderate 3'-5' exonuclease activity and strong strand-displacement. AtPolIs are the first known example of a family-A DNAP evolved to function in both DNA replication and lesion bypass. The lesion bypass capabilities of POPs may be required to prevent replication fork collapse in plant organelles.

| S-EPMC5737093 | biostudies-literature

Ribonucleotide discrimination by translesion synthesis DNA polymerases.

Project description:The well-being of all living organisms relies on the accurate duplication of their genomes. This is usually achieved by highly elaborate replicase complexes which ensure that this task is accomplished timely and efficiently. However, cells often must resort to the help of various additional "specialized" DNA polymerases that gain access to genomic DNA when replication fork progression is hindered. One such specialized polymerase family consists of the so-called "translesion synthesis" (TLS) polymerases; enzymes that have evolved to replicate damaged DNA. To fulfill their main cellular mission, TLS polymerases often must sacrifice precision when selecting nucleotide substrates. Low base-substitution fidelity is a well-documented inherent property of these enzymes. However, incorrect nucleotide substrates are not only those which do not comply with Watson-Crick base complementarity, but also those whose sugar moiety is incorrect. Does relaxed base-selectivity automatically mean that the TLS polymerases are unable to efficiently discriminate between ribonucleoside triphosphates and deoxyribonucleoside triphosphates that differ by only a single atom? Which strategies do TLS polymerases employ to select suitable nucleotide substrates? In this review, we will collate and summarize data accumulated over the past decade from biochemical and structural studies, which aim to answer these questions.

| S-EPMC6261451 | biostudies-literature

Translesion DNA polymerases in eukaryotes: what makes them tick?

Project description:Life as we know it, simply would not exist without DNA replication. All living organisms utilize a complex machinery to duplicate their genomes and the central role in this machinery belongs to replicative DNA polymerases, enzymes that are specifically designed to copy DNA. "Hassle-free" DNA duplication exists only in an ideal world, while in real life, it is constantly threatened by a myriad of diverse challenges. Among the most pressing obstacles that replicative polymerases often cannot overcome by themselves are lesions that distort the structure of DNA. Despite elaborate systems that cells utilize to cleanse their genomes of damaged DNA, repair is often incomplete. The persistence of DNA lesions obstructing the cellular replicases can have deleterious consequences. One of the mechanisms allowing cells to complete replication is "Translesion DNA Synthesis (TLS)". TLS is intrinsically error-prone, but apparently, the potential downside of increased mutagenesis is a healthier outcome for the cell than incomplete replication. Although most of the currently identified eukaryotic DNA polymerases have been implicated in TLS, the best characterized are those belonging to the "Y-family" of DNA polymerases (pols ?, ?, ? and Rev1), which are thought to play major roles in the TLS of persisting DNA lesions in coordination with the B-family polymerase, pol ?. In this review, we summarize the unique features of these DNA polymerases by mainly focusing on their biochemical and structural characteristics, as well as potential protein-protein interactions with other critical factors affecting TLS regulation.

| S-EPMC5573590 | biostudies-literature

Translesion and Repair DNA Polymerases: Diverse Structure and Mechanism.

Project description:The number of DNA polymerases identified in each organism has mushroomed in the past two decades. Most newly found DNA polymerases specialize in translesion synthesis and DNA repair instead of replication. Although intrinsic error rates are higher for translesion and repair polymerases than for replicative polymerases, the specialized polymerases increase genome stability and reduce tumorigenesis. Reflecting the numerous types of DNA lesions and variations of broken DNA ends, translesion and repair polymerases differ in structure, mechanism, and function. Here, we review the unique and general features of polymerases specialized in lesion bypass, as well as in gap-filling and end-joining synthesis.

| S-EPMC6098713 | biostudies-literature

Werner syndrome protein interacts functionally with translesion DNA polymerases.

Project description:Werner syndrome (WS) is characterized by premature onset of age-associated disorders and predisposition to cancer. The WS protein, WRN, encodes 3' --> 5' DNA helicase and 3' --> 5' DNA exonuclease activities, and is implicated in the maintenance of genomic stability. Translesion (TLS) DNA polymerases (Pols) insert nucleotides opposite replication-blocking DNA lesions and presumably prevent replication fork stalling/collapse. Here, we present in vitro and in vivo data that demonstrate functional interaction between WRN and the TLS Pols, Poleta, Polkappa, and Poliota. In vitro, WRN stimulates the extension activity of TLS Pols on lesion-free and lesion-containing DNA templates, and alleviates pausing at stalling lesions. Stimulation is mediated through an increase in the apparent V(max) of the polymerization reaction. Notably, by accelerating the rate of nucleotide incorporation, WRN increases mutagenesis by Poleta. In vivo, WRN and Poleta colocalize at replication-dependent foci in response to UVC irradiation. The functional interaction between WRN and TLS Pols may promote replication fork progression, at the expense of increased mutagenesis, and obviate the need to resolve stalled/collapsed forks by processes involving chromosomal rearrangements.

| S-EPMC1965524 | biostudies-literature

Formation of S-[2-(N6-Deoxyadenosinyl)ethyl]glutathione in DNA and Replication Past the Adduct by Translesion DNA Polymerases.

Project description:1,2-Dibromoethane (DBE, ethylene dibromide) is a potent carcinogen due at least in part to its DNA cross-linking effects. DBE cross-links glutathione (GSH) to DNA, notably to sites on 2'-deoxyadenosine and 2'-deoxyguanosine ( Cmarik , J. L. , et al. ( 1991 ) J. Biol. Chem. 267 , 6672 - 6679 ). Adduction at the N6 position of 2'-deoxyadenosine (dA) had not been detected, but this is a site for the linkage of O6-alkylguanine DNA alkyltransferase ( Chowdhury , G. , et al. ( 2013 ) Angew. Chem. Int. Ed. 52 , 12879 - 12882 ). We identified and quantified a new adduct, S-[2-(N6-deoxyadenosinyl)ethyl]GSH, in calf thymus DNA using LC-MS/MS. Replication studies were performed in duplex oligonucleotides containing this adduct with human DNA polymerases (hPols) ?, ?, and ?, as well as with Sulfolobus solfataricus Dpo4, Escherichia coli polymerase I Klenow fragment, and bacteriophage T7 polymerase. hPols ? and ?, Dpo4, and Klenow fragment were able to bypass the adduct with only slight impedance; hPol ? and ? showed increased misincorporation opposite the adduct compared to that of unmodified 2'-deoxyadenosine. LC-MS/MS analysis of full-length primer extension products by hPol ? confirmed the incorporation of dC opposite S-[2-(N6-deoxyadenosinyl)ethyl]GSH and also showed the production of a -1 frameshift. These results reveal the significance of N6-dA GSH-DBE adducts in blocking replication, as well as producing mutations, by human translesion synthesis DNA polymerases.

| S-EPMC5768145 | biostudies-literature

Translesion synthesis across 1,N2-ethenoguanine by human DNA polymerases.

Project description:1,N(2)-Etheno(epsilon)guanine (epsilon) is formed in DNA as a result of exposure to certain vinyl monomers (e.g., vinyl chloride) or from lipid peroxidation. This lesion has been shown to be mutagenic in bacteria and mammalian cells. 1,N(2)-epsilon-G has been shown to block several model replicative DNA polymerases (pols), with limited bypass. Recently, an archebacterial DNA pol, Sulfolobus solfataricus Dpo4, has been shown to copy past 1,N(2)-epsilon-G. In this study, we examined the abilities of recombinant, full-length human pol delta and three human translesion DNA pols to copy past 1,N(2)-epsilon-G. The replicative pol, pol delta, was completely blocked. Pols iota and kappa showed similar rates of incorporation of dTTP and dCTP. Pol eta was clearly the most active of these pols in copying past 1,N(2)-epsilon-G, incorporating in the order dGTP > dATP > dCTP, regardless of whether the base 5' of 1,N(2)-epsilon-G in the template was C or T. Pol eta also had the highest error frequency opposite 1,N(2)-epsilon-G. Analysis of the extended products of the pol eta reactions by mass spectrometry indicated only two products, both of which had G incorporated opposite 1,N(2)-epsilon-G and all other base pairing being normal (i.e., G:C and A:T). One-half of the products contained an additional A at the 3'-end, presumably arising from a noninformational blunt end addition or possibly a slipped insertion mechanism at the end of the primer-template replication process. In summary, the most efficient of the four human DNA pols was pol eta, which appeared to insert G opposite 1,N(2)-epsilon-G and then copy correctly. This pattern differs with the same oligonucleotide sequences and 1,N(2)-epsilon-G observed using Dpo4, emphasizing the importance of pols in mutagenesis events.

| S-EPMC3130186 | biostudies-literature

Sequential assembly of translesion DNA polymerases at UV-induced DNA damage sites.

Project description:In response to DNA damage such as from UV irradiation, mammalian Y-family translesion synthesis (TLS) polymerases Pol? and Rev1 colocalize with proliferating cell nuclear antigen at nuclear foci, presumably representing stalled replication sites. However, it is unclear whether the localization of one polymerase is dependent on another. Furthermore, there is no report on the in vivo characterization of the Rev3 catalytic subunit of the B-family TLS polymerase Pol?. Here we describe the detection of endogenous human Pol?, Rev1, and Rev3 by immunocytochemistry using existing or newly created antibodies, as well as various means of inhibiting their expression, which allows us to examine the dynamics of endogenous TLS polymerases in response to UV irradiation. It is found that Rev1 and Pol? are independently recruited to the nuclear foci, whereas the Rev3 nuclear focus formation requires Rev1 but not Pol?. In contrast, neither Rev1 nor Pol? recruitment requires Rev3. To further support these conclusions, we find that simultaneous suppression of Pol? and Rev3 results in an additive cellular sensitivity to UV irradiation. These observations suggest a cooperative and sequential assembly of TLS polymerases in response to DNA damage. They also support and extend the current polymerase switch model.

| S-EPMC3128538 | biostudies-literature

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