TENT4A Non-Canonical Poly(A) Polymerase Regulates DNA-Damage Tolerance via Multiple Pathways That Are Mutated in Endometrial Cancer.

TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here, we show that TENT4A regulates multiple biological pathways and focuses on its multilayer regulation of translesion DNA synthesis (TLS), in which error-prone DNA polymerases bypass unrepaired DNA lesions. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase η and RAD18 E3 ligase, which guides the polymerase to replication stalling sites and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase η, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.

Mutational signatures reveal the role of RAD52 in p53-independent p21-driven genomic instability.

BACKGROUND: Genomic instability promotes evolution and heterogeneity of tumors. Unraveling its mechanistic basis is essential for the design of appropriate therapeutic strategies. In a previous study, we reported an unexpected oncogenic property of p21WAF1/Cip1, showing that its chronic expression in a p53-deficient environment causes genomic instability by deregulation of the replication licensing machinery. RESULTS: We now demonstrate that p21WAF1/Cip1 can further fuel genomic instability by suppressing the repair capacity of low- and high-fidelity pathways that deal with nucleotide abnormalities. Consequently, fewer single nucleotide substitutions (SNSs) occur, while formation of highly deleterious DNA double-strand breaks (DSBs) is enhanced, crafting a characteristic mutational signature landscape. Guided by the mutational signatures formed, we find that the DSBs are repaired by Rad52-dependent break-induced replication (BIR) and single-strand annealing (SSA) repair pathways. Conversely, the error-free synthesis-dependent strand annealing (SDSA) repair route is deficient. Surprisingly, Rad52 is activated transcriptionally in an E2F1-dependent manner, rather than post-translationally as is common for DNA repair factor activation. CONCLUSIONS: Our results signify the importance of mutational signatures as guides to disclose the repair history leading to genomic instability. We unveil how chronic p21WAF1/Cip1 expression rewires the repair process and identifies Rad52 as a source of genomic instability and a candidate therapeutic target.

High-resolution genomic assays provide insight into the division of labor between TLS and HDR in mammalian replication of damaged DNA.

The multitude of DNA lesions that continuously form in DNA cannot all be detected and removed prior to replication. Thus, encounters of the replication fork with DNA damage become inevitable. Such encounters inhibit fork progression, leading to replication fork arrest or to replication re-priming downstream of the damage site. Either of these events will result in the formation of gap-lesion structures, in which a damaged base is located in a single stranded stretch of DNA, that is vulnerable to subsequent nicking. The double strand break that would ensue if ssDNA becomes nicked constitutes escalation of the damage from nucleotide(s)-specific to chromosomal scale. Cells employ two universal DNA damage tolerance (DDT) strategies to resolve these situations, by converting the gap-lesion structures into dsDNA without repairing the damage. The first is translesion DNA synthesis (TLS), in which a specialized low-fidelity DNA polymerase inserts a nucleotide opposite the damaged one. TLS is inherently mutagenic, due to the miscoding nature of most damaged nucleotides. The second strategy is homology-dependent repair (HDR), which relies on the presence of an identical intact sister chromatid. The molecular mechanisms that regulate the division of labor between these pathways are poorly understood. This review focuses on the balance between TLS and HDR in mammalian cells, discussing recent findings that were made possible thanks to newly developed high resolution genomic assays, and highlighting the role of the DNA lesion's properties in DDT pathway choice.

Dietary Calorie Restriction from Adulthood Through Old Age in Rats: Improved DNA Polymerase ß and DNA Gap Repair Activity in Cortical Neurons.

It is well established now that dietary calorie restriction (CR) leads to extension of life span in many species, although the exact mechanism of this effect is still eluding. In the present study, we examined the effect of 40 % CR imposed during a prolonged period of life span (from 6 to 30 months) of rats on the activity of DNA polymerase ß (pol ß) in view of its role in short gap base excision DNA repair and template driven primer extension. DNA pol ß activity is very low at this late age. However, cortical neuronal extracts prepared from CR rats of 30 months age showed significantly higher pol ß protein levels and activity when compared to control 30 month old rats. Yet, one-nucleotide gap repair in old control neurons and an improved efficiency in CR neurons could be visualized only after supplementation of the extracts with T4 DNA ligase indicating the lack of CR affect on ligase activity. No impressive primer extension activity is seen either in the CR or old control neurons. These results are taken to convey that extended CR through adult life leads to improved pol ß activity and therefore, pol ß dependent DNA gap repair activity.

Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin.

Cells cope with replication-blocking lesions via translesion DNA synthesis (TLS). TLS is carried out by low-fidelity DNA polymerases that replicate across lesions, thereby preventing genome instability at the cost of increased point mutations. Here we perform a two-stage siRNA-based functional screen for mammalian TLS genes and identify 17 validated TLS genes. One of the genes, NPM1, is frequently mutated in acute myeloid leukaemia (AML). We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-η (polη), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of polη. Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in ~30% of AML patients results in excessive degradation of polη. These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1.

Topoisomerase IIß regulates base excision repair capacity of neurons.

Topoisomerase IIß (TopoIIß), an enzyme involved in DNA rearrangements, is predominantly present in brain and its levels are shown to decrease with age. This study characterizes the function of TopoIIß in regulating BER (base excision repair) activity. TopoIIß deficient granule neurons (CGNT⁻) show greater sensitivity to N-ethyl N-nitroso urea (ENU)-mediated DNA damage. The cell-free extracts of TopoIIß knockdown cells (ECGNT⁻) show a significant decrease in G-U BER activity during ENU-treatment as well as during recovery, suggesting that TopoIIß promotes G-U BER activity. Since G-U BER activity is not affected in the presence of ICRF-193, catalytic inhibitor of TopoIIß, the activity of enzyme per se may not be participating in BER activity. Further characterization of the activities of BER enzymes present in ECGNT⁻ shows that uracil DNA-glycosylase (UDG) and ligase (LIG) activities decrease significantly in both ENU treatment and recovery. Supplementation of TopoIIß to ECGNT⁻ does not restore ligation activity and ICRF-193 does not influence the LIG activity. These results suggest a role, at least an indirect one, of TopoIIß in the repair of ENU-mediated strand breaks via BER pathway including the activities of UDG and LIG.

Age-dependent decline of DNA base excision repair activity in rat cortical neurons.

Synthetic oligonucleotide duplexes containing a single uracil (U) or 8-oxoguanine (8-oxoG) were used as a model substrates to assess the base excision repair (BER) ability of neuronal extracts prepared from the cerebral cortex of young (7 days), adult (180 days) and old (720 days) rats. Our results demonstrate that BER activity in neurons markedly declines with age. The decline in BER could be attributed to decrease in the expression levels and activities of BER enzymes. Supplementing neuronal extracts with uracil DNA-glycosylase (UDG), 8-oxoguanine DNA glycosylase (OGG1), apurinic endonuclease 1, pol ß and T(4) DNA ligase independently could not restore the loss of BER activity in adult and old neuronal extracts. However, supplementation of pol ß in combination of T(4) DNA ligase to neuronal extract, improved the BER in adult and old neuronal extracts. Additional supplementation of the extracts with UDG or OGG1 apart from pol ß and T(4) DNA ligase, or with all pure enzymes restored very markedly the loss of BER in aging neurons. These results suggest the age-dependent decline in BER is due to an overall deficiency of the various factors needed for BER but pol ß and DNA ligase seem to be the most limiting factors.

Age-related decline in DNA polymerase ß activity in rat brain and tissues.

Fidelity of DNA polymerases is vital for maintaining genomic integrity. Deficient DNA repair leads to age related disorders or cancer. If the age at which the decline in activity of predominant DNA repair enzymes starts is identified, and the deficient proteins supplemented, then the manifestation of these diseases can be delayed promoting healthy aging. DNA polymerase ß (pol ß) is a predominant repair enzyme in brain. DNA pol ß activity declines with age in rat brain/neurons but the exact age during the life time of rat when this decline begins is not known, and comparison of this activity was not made between post mitotic and proliferating tissues therefore the pattern of pol ß with age was studied in rat brain and tissues. The decline in pol ß activity started between 30 and 45 days postnatal in all the tissues. Post mitotic tissues showed pronounced decline than the proliferating tissues.

Studies on the molecular correlates of genomic stability in rat brain cells following Amalakirasayana therapy.

Adult Wistar NIN (WNIN) rats (6 months old) of both sexes were orally fed Amalakirasayana at a dose of 4.5 g per kg body weight, five days in a week. The Amalakirasayana was prepared by Arya Vaidya Sala, Kottakkal, Kerala, India, which is considered as gold standard. After 3, 9 and 15 months of such therapeutic regime, rats were sacrificed and various tissues including brain were removed. Isolated cell suspensions of neurons and astroglia were prepared from the cerebral cortex. DNA damage, as a prime indicator of the status of genomic stability was measured in terms of single (SSBs) and double strand breaks (DSBs) through (a). The "comet" assay and (b). The biochemical methods utilizing the unique properties of Escherichia coli DNA polymerase I (pol I) and calf thymus terminal transferase. The results convincingly indicate that while in control animals, there was a distinct increase in DNA damage with age in neurons and astrocytes, rasayana fed animals showed significantly less DNA damage in brain cells demonstrating beneficial effects of Rasayana therapy towards maintenance of genomic stability. DNA-damage may be the proximal cause of aging and strategies to reduce the rate of aging could be based on this fact.

Study of DNA damage via the comet assay and base excision repair activities in rat brain neurons and astrocytes during aging.

Earlier we have used biochemical approach to assess the number of single (SSBs) and double (DSBs) strand breaks in brain cellular DNA. However, a quick method to obtain a reliable measure of DNA damage in cells was in need for population studies. Therefore, single cell gel electrophoresis technique (popularly known as "comet" assay) has been standardized using the Trevigen protocol. DNA damage was assessed in isolated neurons and astrocytes from the cortex of young (7 days), adult (6 months) and old (2 years). Marked increase is seen in DNA damage in terms SSBs and DSBs in both types of cells by 6 months of age, which increased further by 2 years of age. The number of 8-oxoguanine DNA glycosylase (OGG1) and uracil DNA glycosylase (UDG) sensitive sites also increased in DNA with age with the simultaneous decrease in OGG1, UDG and AP endonuclease (APE1) activities. Thus the comet assay adapted to our lab conditions has proven to be useful for a quick assessment of DNA damage in a large number of samples that constitute our future studies.

Cell death is associated with reduced base excision repair during chronic alcohol administration in adult rat brain.

The cell death cascades in different brain regions namely hippocampus and frontal cortex of rats fed with 10% (v/v) ethanol for 12 weeks, was examined. After Western blotting, different cell death associated proteins displayed differential activation in the two regions observed. In hippocampus, activated caspase-3 and caspase-7 resulted in subsequent cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). Cytochrome c release to cytosol and apoptosis inducing factor (AIF) translocation to nucleus was marginal. B-cell leukemia/lymphoma-2 (Bcl-2) translocation to cytosol was significant whereas Bcl-2-associated X protein (Bax) and Bcl-associated death protein (Bad) were largely located in cytosol. Further, upregulation of N-methyl D-aspartate receptor subunit 1 (NMDAR1), N-methyl D-aspartate receptor subunit 2B (NMDAR2B), N-methyl D-aspartate receptor subunit 2C (NMDAR2C) and activation of calpains were observed. In frontal cortex, caspase-3 activation, cleavage of PARP-1 and nuclear translocation of AIF were more pronounced. Moreover, cytochrome c release to cytosol, Bcl-2 translocation to cytosol was evident. However, levels of Bax, Bad, NMDA receptor subunits, and calpains were unaffected. Apoptosis was further substantiated by in situ staining for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). Results of the current study revealed that frontal cortex exhibits a higher level of ethanol-induced apoptosis relative to hippocampus. DNA polymerase beta assay and immunoblot showed significant loss in base excision repair in ethanol treated group.