Novel roles of DNA damage repair enzymes in the processing of modified ribonucleotides embedded in DNA

From data in literature, it has well known that 100 million rNMPs are transiently present in mammalian DNA for several reasons. Moreover, the presence of RNA as single or more ribonucleotides into DNA results very dangerous for the cell because able to distort the double helix DNA. A specific pathway acts in order to remove this lesion called Ribonucleotide Excision Repair (RER) pathway in which RNase H2 has an important role as endonuclease, able to cleave at the 5\u2019 side of rNMP in DNA. Although in last decade, huge steps forward have been done in this field, more studies are needed for better understanding the impact of this lesion on DNA and their back-up repair mechanism when RER does not work, as happens in several pathologies including cancer and Aicardi-Goutieres syndrome. Because of the high abundance of rNMPs in DNA and the ability of several polymerases to insert and elongate oxidized rGMP during DNA replication, the hypothesis of the incorporation of modified rNMPs (including abasic and oxidized rNMPs such as 7,8-dihydro-8-oxo-riboguanosine) results feasible. Moreover, these data underscore the possibility, not only regarding the presence of modified rNMPs into DNA, but also the necessity to determine how cells can target and remove oxidized or abasic rNMPs from DNA. Until now, nothing is known about the putative role of Base Excision Repair (BER) pathway in the removal of unmodified and modified rNMPs embedded in DNA. APE1 is the most important protein that works in BER pathway as endonuclease, able to cleave 5\u2032 side of deoxyabasic sites generated spontaneously or following the processing of glycosylases (including OGG1) on a damaged (including oxidized) base. Moreover, APE1 has nucleotide incision repair activity on different modified bases, which are directly repaired by APE1 bypassing the action of specific glycosylases. Finally BER, but specifically APE1, has an important involvement in RNA metabolism and RNA-decay as demonstrated by its ability to cleave abasic single-stranded RNA and for having 3\u2032-RNA phosphatase and weak 3\u2032-5\u2032 exoribonuclease activities. For this reason, there is a high likelihood that BER enzymes could be involved in the processing of rNMPs in DNA, particularly in the case of chemically modified rNMPs, such as abasic and oxidized rNMPs. Therefore, we focused our attention on enzymes belonging to RER and BER enzymes in order to investigate: If BER can be work as a back up repair mechanism when RER is inefficient; If RER is involved in the processing of abasic and oxidized rNMPs embedded in DNA; and if not, if BER is involved in the processing of abasic and oxidized rNMPs embedded in DNA. For all the experiments, we have used different DNA oligonucleotides in which an abasic or an oxidized ribonucleotide was embedded in the middle of the sequence. First, we tested recombinant and cell-free extracts RNase H2. Surprisingly, we discovered that RNase H2, although able to process normal rG embedded in DNA, is not able to cleave abasic and oxidized rNMPs embedded in DNA leaving the hypothesis that RER can not work on this lesion. After that, we moved on BER pathway, specifically on APE1 and OGG1 proteins. We discovered that APE1, as 5\u2019-endonuclease, does not cleave unmodified rNMPs embedded in DNA. So, APE1 may not work as back up when RNase H2 is inefficient as happens for different pathologies including cancer and Aicardi-Goutieres syndrome. We discovered and characterized an unknown APE1 activity on abasic ribonucleotide embedded in DNA. We then analyzed the activities of 8-oxoguanine DNA glycosylase (OGG1) and APE1 to recognize and cleave an oxidized rNMP. Our data demonstrate that OGG1, although able to bind the oxidized rG, is inefficient in the cleavage of this substrate. Surprisingly, APE1 shows a weak endoribonuclease activity on the oxidized substrate, maybe associable to a putative NIR activity of APE1. All these results show a strong impact on the DNA repair field.From data in literature, it has well known that 100 million rNMPs are transiently present in mammalian DNA for several reasons. Moreover, the presence of RNA as single or more ribonucleotides into DNA results very dangerous for the cell because able to distort the double helix DNA. A specific pathway acts in order to remove this lesion called Ribonucleotide Excision Repair (RER) pathway in which RNase H2 has an important role as endonuclease, able to cleave at the 5\u2019 side of rNMP in DNA. Although in last decade, huge steps forward have been done in this field, more studies are needed for better understanding the impact of this lesion on DNA and their back-up repair mechanism when RER does not work, as happens in several pathologies including cancer and Aicardi-Goutieres syndrome. Because of the high abundance of rNMPs in DNA and the ability of several polymerases to insert and elongate oxidized rGMP during DNA replication, the hypothesis of the incorporation of modified rNMPs (including abasic and oxidized rNMPs such as 7,8-dihydro-8-oxo-riboguanosine) results feasible. Moreover, these data underscore the possibility, not only regarding the presence of modified rNMPs into DNA, but also the necessity to determine how cells can target and remove oxidized or abasic rNMPs from DNA. Until now, nothing is known about the putative role of Base Excision Repair (BER) pathway in the removal of unmodified and modified rNMPs embedded in DNA. APE1 is the most important protein that works in BER pathway as endonuclease, able to cleave 5\u2032 side of deoxyabasic sites generated spontaneously or following the processing of glycosylases (including OGG1) on a damaged (including oxidized) base. Moreover, APE1 has nucleotide incision repair activity on different modified bases, which are directly repaired by APE1 bypassing the action of specific glycosylases. Finally BER, but specifically APE1, has an important involvement in RNA metabolism and RNA-decay as demonstrated by its ability to cleave abasic single-stranded RNA and for having 3\u2032-RNA phosphatase and weak 3\u2032-5\u2032 exoribonuclease activities. For this reason, there is a high likelihood that BER enzymes could be involved in the processing of rNMPs in DNA, particularly in the case of chemically modified rNMPs, such as abasic and oxidized rNMPs. Therefore, we focused our attention on enzymes belonging to RER and BER enzymes in order to investigate: If BER can be work as a back up repair mechanism when RER is inefficient; If RER is involved in the processing of abasic and oxidized rNMPs embedded in DNA; and if not, if BER is involved in the processing of abasic and oxidized rNMPs embedded in DNA. For all the experiments, we have used different DNA oligonucleotides in which an abasic or an oxidized ribonucleotide was embedded in the middle of the sequence. First, we tested recombinant and cell-free extracts RNase H2. Surprisingly, we discovered that RNase H2, although able to process normal rG embedded in DNA, is not able to cleave abasic and oxidized rNMPs embedded in DNA leaving the hypothesis that RER can not work on this lesion. After that, we moved on BER pathway, specifically on APE1 and OGG1 proteins. We discovered that APE1, as 5\u2019-endonuclease, does not cleave unmodified rNMPs embedded in DNA. So, APE1 may not work as back up when RNase H2 is inefficient as happens for different pathologies including cancer and Aicardi-Goutieres syndrome. We discovered and characterized an unknown APE1 activity on abasic ribonucleotide embedded in DNA. We then analyzed the activities of 8-oxoguanine DNA glycosylase (OGG1) and APE1 to recognize and cleave an oxidized rNMP. Our data demonstrate that OGG1, although able to bind the oxidized rG, is inefficient in the cleavage of this substrate. Surprisingly, APE1 shows a weak endoribonuclease activity on the oxidized substrate, maybe associable to a putative NIR activity of APE1. All these results show a strong impact on the DNA repair field

Platinum Salts in Patients with Breast Cancer: A Focus on Predictive Factors

Breast cancer (BC) is the most frequent oncologic cause of death among women and the improvement of its treatments is compelling. Platinum salts (e.g., carboplatin, cisplatin, and oxaliplatin) are old drugs still used to treat BC, especially the triple-negative subgroup. However, only a subset of patients see a concrete benefit from these drugs, raising the question of how to select them properly. Therefore, predictive biomarkers for platinum salts in BC still represent an unmet clinical need. Here, we review clinical and preclinical works in order to summarize the current evidence about predictive or putative platinum salt biomarkers in BC. The association between BRCA1/2 gene mutations and platinum sensitivity has been largely described. However, beyond the mutations of these two genes, several other proteins belonging to the homologous recombination pathways have been linked to platinum response, defining the concept of BRCAness. Several works, here reviewed, have tried to capture BRCAness through different strategies, such as homologous recombination deficiency (HRD) score and genetic signatures. Moreover, p53 and its family members (p63 and p73) might also be used as predictors of platinum response. Finally, we describe the mounting preclinical evidence regarding base excision repair deficiency as a possible new platinum biomarker

Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2

Ribonucleoside 5'-monophosphates (rNMPs) are the most common non-standard nucleotides found in DNA of eukaryotic cells, with over 100 million rNMPs transiently incorporated in the mammalian genome per cell cycle. Human ribonuclease (RNase) H2 is the principal enzyme able to cleave rNMPs in DNA. Whether RNase H2 may process abasic or oxidized rNMPs incorporated in DNA is unknown. The base excision repair (BER) pathway is mainly responsible for repairing oxidized and abasic sites into DNA. Here we show that human RNase H2 is unable to process an abasic rNMP (rAP site) or a ribose 8oxoG (r8oxoG) site embedded in DNA. On the contrary, we found that recombinant purified human apurinic/apyrimidinic endonuclease-1 (APE1) and APE1 from human cell extracts efficiently process an rAP site in DNA and have weak endoribonuclease and 3'-exonuclease activities on r8oxoG substrate. Using biochemical assays, our results provide evidence of a human enzyme able to recognize and process abasic and oxidized ribonucleotides embedded in DNA

Inhibition of APE1-endonuclease activity affects cell metabolism in colon cancer cells via a p53-dependent pathway

The pathogenesis of colorectal cancer (CRC) involves different mechanisms, such as genomic and microsatellite instabilities. Recently, a contribution of the base excision repair (BER) pathway in CRC pathology has been emerged. In this context, the involvement of APE1 in the BER pathway and in the transcriptional regulation of genes implicated in tumor progression strongly correlates with chemoresistance in CRC and in more aggressive cancers. In addition, the APE1 interactome is emerging as an important player in tumor progression, as demonstrated by its interaction with Nucleophosmin (NPM1). For these reasons, APE1 is becoming a promising target in cancer therapy and a powerful prognostic and predictive factor in several cancer types. Thus, specific APE1 inhibitors have been developed targeting: i) the endonuclease activity; ii) the redox function and iii) the APE1-NPM1 interaction. Furthermore, mutated p53 is a common feature of advanced CRC. The relationship between APE1 inhibition and p53 is still completely unknown. Here, we demonstrated that the inhibition of the endonuclease activity of APE1 triggers p53-mediated effects on cell metabolism in HCT-116 colon cancer cell line. In particular, the inhibition of the endonuclease activity, but not of the redox function or of the interaction with NPM1, promotes p53 activation in parallel to sensitization of p53-expressing HCT-116 cell line to genotoxic treatment. Moreover, the endonuclease inhibitor affects mitochondrial activity in a p53-dependent manner. Finally, we demonstrated that 3D organoids derived from CRC patients are susceptible to APE1-endonuclease inhibition in a p53-status correlated manner, recapitulating data obtained with HCT-116 isogenic cell lines. These findings suggest the importance of further studies aimed at testing the possibility to target the endonuclease activity of APE1 in CRC

Expression of APE1 endonuclease in primarily cultured aortic valve interstitial cells undergoing spontaneous senescence

Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is the major enzyme involved in the base excision repair pathway working on DNA damages mainly caused by oxidative stress. Namely, APE1 hydrolyses the phosphodiester bond at the abasic sites generated by DNA glycosylases, creating the substrate for DNA polymerase β and DNA ligase IIIa which terminate the reparative process [1]. APE1 also seems to play a role in cell senescence maintaining telomere stability and size in interaction with specifi c telomere-protective proteins [2]. Here, aortic valve interstitial cells (AVICs) isolated from healthy bovine valve leafl ets were cultured under normal conditions for up to 90 days to achieve spontaneous cell senescence. Time-dependent increase in β-galactosidase activity, a marker of cell senescence, was paralleled by a remarkable decrease of APE1-expressing AVICs starting from day 60, as immunocytochemically revealed. Quantitative Western blot analyses also showed a drop of APE1 protein content at day 60, whereas RTPCR analyses revealed a mild increase of the enzyme mRNA over time. Ultrastructurally, AVICs appeared well preserved up to 30-day-long culturing. Conversely, starting from day 60, cells showed non-lysosomal autophagocytosis features mainly consisting of a hypertrophic rough endoplasmic reticulum engulfi ng suff ering mitochondria [3]. Cytoplasm vacuolization due to large organelle degeneration was also clearly appreciable. In conclusion, decreasing APE1 expression over time is supposed to contribute to AVIC decay in a model of spontaneous cell senescence

Human AP-endonuclease (Ape1) activity on telomeric G4 structures is modulated by acetylatable lysine residues in the N-terminal sequence

Loss of telomeres stability is a hallmark of cancer cells. Exposed telomeres are prone to aberrant end joining reactions leading to chromosomal fusions and translocations. Human telomeres contain repeated TTAGGG elements, in which the 3' exposed strand may adopt a G-quadruplex (G4) structure. The guanine-rich regions of telomeres are hotspots for oxidation forming 8-oxoguanine, a lesion that is handled by the base excision repair (BER) pathway. One key player of this pathway is Ape1, the main human endonuclease processing abasic sites. Recent evidences showed an important role for Ape1 in telomeric physiology, but the molecular details regulating Ape1 enzymatic activities on G4-telomeric sequences are lacking. Through a combination of in vitro assays, we demonstrate that Ape1 can bind and process different G4 structures and that this interaction involves specific acetylatable lysine residues (i.e. K-27/31/32/35) present in the unstructured N-terminal sequence of the protein. The cleavage of an abasic site located in a G4 structure by Ape1 depends on the DNA conformation or the position of the lesion and on electrostatic interactions between the protein and the nucleic acids. Moreover, Ape1 mutants mimicking the acetylated protein display increased cleavage activity for abasic sites. We found that nucleophosmin (NPM1), which binds the N-terminal sequence of Ape1, plays a role in modulating telomere length and Ape1 activity at abasic G4 structures. Thus, the Ape1 N-terminal sequence is an important relay site for regulating the enzyme's activity on G4-telomeric sequences, and specific acetylatable lysine residues constitute key regulatory sites of Ape1 enzymatic activity dynamics at telomeres

Unlike the Escherichia coli counterpart, archaeal RNase HII cannot process ribose monophosphate abasic sites and oxidized ribonucleotides embedded in DNA

The presence of ribonucleoside monophosphates (rNMPs) in nuclear DNA decreases genome stability. To ensure survival despite rNMP insertions, cells have evolved a complex network of DNA repair mechanisms, in which the ribonucleotide excision repair pathway, initiated by type 2 ribonuclease H (RNase HII/2), plays a major role. We recently demonstrated that eukaryotic RNase H2 cannot repair damaged, that is, ribose monophosphate abasic (both apurinic or apyrimidinic) site (rAP) or oxidized rNMP embedded in DNA. Currently, it remains unclear why RNase H2 is unable to repair these modified nucleic acids having either only a sugar moiety or an oxidized base. Here, we compared the endoribonuclease specificity of the RNase HII enzymes from the archaeon Pyrococcus abyssi and the bacterium Escherichia coli, examining their ability to process damaged rNMPs embedded in DNA in vitro. We found that E. coli RNase HII cleaves both rAP and oxidized rNMP sites. In contrast, like the eukaryotic RNase H2, P. abyssi RNase HII did not display any rAP or oxidized rNMP incision activities, even though it recognized them. Notably, the archaeal enzyme was also inactive on a mismatched rNMP, whereas the E. coli enzyme displayed strong preference for the mispaired rNMP over the paired rNMP in DNA. On the basis of our biochemical findings and also structural modeling analyses of RNase HII/2 proteins from organisms belonging to all three domains of life, we propose that RNases HII/2’s dual roles in RER and RNA/DNA hydrolysis result in limited acceptance of modified rNMPs embedded in DNA