Error-free bypass of 2-hydroxyadenine by human DNA polymerase λ with Proliferating Cell Nuclear Antigen and Replication Protein A in different sequence contexts

1,2-dihydro-2-oxoadenine (2-OH-A), a common DNA lesion produced by reactive oxygen species, is a strong replicative block for several DNA polymerases (DNA pols). We have previously shown that various bases can be misincorporated opposite the 2-OH-A lesion and the type of mispairs varies with either the sequence context or the type of DNA pol tested. Here, we have analysed the ability of the human pol family X member DNA pol λ, to bypass the 2-OH-A lesion. DNA pol λ can perform error-free bypass of 2-OH-A when this lesion is located in a random sequence, whereas in a repeated sequence context, even though bypass was also largely error-free, misincorporation of dGMP could be observed. The fidelity of translesion synthesis of 2-OH-A in a repeated sequence by DNA pol λ was enhanced by the auxiliary proteins Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RP-A). We also found that the DNA pol λ active site residue tyrosine 505 determined the nucleotide selectivity opposite 2-OH-A. Our data show, for the first time, that the 2-OH-A lesion can be efficiently and faithfully bypassed by a human DNA pol λ in combination with PCNA and RP-A

Error-free bypass of 2-hydroxyadenine by human DNA polymerase λ with Proliferating Cell Nuclear Antigen and Replication Protein A in different sequence contexts

1,2-dihydro-2-oxoadenine (2-OH-A), a common DNA lesion produced by reactive oxygen species, is a strong replicative block for several DNA polymerases (DNA pols). We have previously shown that various bases can be misincorporated opposite the 2-OH-A lesion and the type of mispairs varies with either the sequence context or the type of DNA pol tested. Here, we have analysed the ability of the human pol family X member DNA pol λ, to bypass the 2-OH-A lesion. DNA pol λ can perform error-free bypass of 2-OH-A when this lesion is located in a random sequence, whereas in a repeated sequence context, even though bypass was also largely error-free, misincorporation of dGMP could be observed. The fidelity of translesion synthesis of 2-OH-A in a repeated sequence by DNA pol λ was enhanced by the auxiliary proteins Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RP-A). We also found that the DNA pol λ active site residue tyrosine 505 determined the nucleotide selectivity opposite 2-OH-A. Our data show, for the first time, that the 2-OH-A lesion can be efficiently and faithfully bypassed by a human DNA pol λ in combination with PCNA and RP-

Microhomology-mediated DNA strand annealing and elongation by human DNA polymerases λ and β on normal and repetitive DNA sequences

‘Classical' non-homologous end joining (NHEJ), dependent on the Ku70/80 and the DNA ligase IV/XRCC4 complexes, is essential for the repair of DNA double-strand breaks. Eukaryotic cells possess also an alternative microhomology-mediated end-joining (MMEJ) mechanism, which is independent from Ku and DNA ligase 4/XRCC4. The components of the MMEJ machinery are still largely unknown. Family X DNA polymerases (pols) are involved in the classical NHEJ pathway. We have compared in this work, the ability of human family X DNA pols β, λ and μ, to promote the MMEJ of different model templates with terminal microhomology regions. Our results reveal that DNA pol λ and DNA ligase I are sufficient to promote efficient MMEJ repair of broken DNA ends in vitro, and this in the absence of auxiliary factors. However, DNA pol β, not λ, was more efficient in promoting MMEJ of DNA ends containing the (CAG)n triplet repeat sequence of the human Huntingtin gene, leading to triplet expansion. The checkpoint complex Rad9/Hus1/Rad1 promoted end joining by DNA pol λ on non-repetitive sequences, while it limited triplet expansion by DNA pol β. We propose a possible novel role of DNA pol β in MMEJ, promoting (CAG)n triplet repeats instabilit

Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases β and λ

We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) λ and β. Here we present the identification of novel non-nucleoside triphosphate (NNTP) derivatives belonging to three classes: (i) non-substrate-specific inhibitors of DNA pol λ; (ii) substrate inhibitors which could preferentially be incorporated by either DNA pol λ wild type or its Y505A mutant and (iii) the substrate inhibitor N-(Biphenylcarbonyl)-4-oxobutyl triphosphate which could be incorporated exclusively by DNA pol β in a Mg(2+)-dependent manner, and preferentially pairs with A on the template. This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases. Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrates

Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases β and λ

We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) λ and β. Here we present the identification of novel non-nucleoside triphosphate (NNTP) derivatives belonging to three classes: (i) non-substrate-specific inhibitors of DNA pol λ; (ii) substrate inhibitors which could preferentially be incorporated by either DNA pol λ wild type or its Y505A mutant and (iii) the substrate inhibitor N-(Biphenylcarbonyl)-4-oxobutyl triphosphate which could be incorporated exclusively by DNA pol β in a Mg2+-dependent manner, and preferentially pairs with A on the template. This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases. Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrate

Identification and Biological Characterization of the Pyrazolo[3,4-d]pyrimidine Derivative SI388 Active as Src Inhibitor

Src is a non-receptor tyrosine kinase (TK) whose involvement in cancer, including glioblastoma (GBM), has been extensively demonstrated. In this context, we started from our in-house library of pyrazolo[3,4-d]pyrimidines that are active as Src and/or Bcr-Abl TK inhibitors and performed a lead optimization study to discover a new generation derivative that is suitable for Src kinase targeting. We synthesized a library of 19 compounds, 2a-s. Among these, compound 2a (SI388) was identified as the most potent Src inhibitor. Based on the cell-free results, we investigated the effect of SI388 in 2D and 3D GBM cellular models. Interestingly, SI388 significantly inhibits Src kinase, and therefore affects cell viability, tumorigenicity and enhances cancer cell sensitivity to ionizing radiation

Incorporation of non-nucleoside triphosphate analogues opposite to an abasic site by human DNA polymerases β and λ

A novel class of non-nucleoside triphosphate analogues, bearing hydrophobic groups sterically similar to nucleosides linked to the α-phosphate but lacking the chemical functional groups of nucleic acids, were tested against six different DNA polymerases (polymerases). Human polymerases α, β and λ, and Saccharomyces cerevisiae polymerase IV, were inhibited with different potencies by these analogues. On the contrary, Escherichia coli polymerase I and HIV-1 reverse transcriptase were not. Polymerase β incorporated these derivatives in a strictly Mn++-dependent manner. On the other hand, polymerase λ could incorporate some alkyltriphosphate derivatives with both Mg++ and Mn++, but only opposite to an abasic site on the template strand. The active site mutant polymerase λ Y505A showed an increased ability to incorporate the analogues. These results show for the first time that neither the base nor the sugar moieties of nucleotides are required for incorporation by family X DNA polymerase

Indolyl aryl sulphones as HIV-1 non-nucleoside reverse transcriptase inhibitors: synthesis, biological evaluation and binding mode studies of new derivatives at indole-2-carboxamide.

New non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are active against the commonly occurring mutations of HIV are urgently needed for the treatment of AIDS. We synthesized new NNRTIs of the indolyl aryl sulphone (IAS) family, which are endowed with high antiviral potency against HIV-1 wt (wild-type), and the Y181C and K103N-Y181C drug resistant mutant strains. Several new compounds were highly active in lymphocytes infected with primary isolates carrying the K103N-V108I-M184V and L100I-V108I mutations. The design of new IASs was based on three-dimensional quantitative structure-activity relationship (3D QSAR) studies and docking simulations. A cross-docking study was also undertaken to gain some insights in to the binding mode of the newly synthesized IASs in the wt and mutated isoforms of reverse transcriptase

Incorporation of non-nucleoside triphosphate analogues opposite to an abasic site by human DNA polymerases β and λ

A novel class of non-nucleoside triphosphate analogues, bearing hydrophobic groups sterically similar to nucleosides linked to the α-phosphate but lacking the chemical functional groups of nucleic acids, were tested against six different DNA polymerases (polymerases). Human polymerases α, β and λ, and Saccharomyces cerevisiae polymerase IV, were inhibited with different potencies by these analogues. On the contrary, Escherichia coli polymerase I and HIV-1 reverse transcriptase were not. Polymerase β incorporated these derivatives in a strictly Mn(++)-dependent manner. On the other hand, polymerase λ could incorporate some alkyltriphosphate derivatives with both Mg(++) and Mn(++), but only opposite to an abasic site on the template strand. The active site mutant polymerase λ Y505A showed an increased ability to incorporate the analogues. These results show for the first time that neither the base nor the sugar moieties of nucleotides are required for incorporation by family X DNA polymerases

Chiral indolylarylsulfone non-nucleoside reverse transcriptase inhibitors as new potent and broad spectrum anti-HIV-1 agents

We designed and synthesized a series of chiral indolyarylsulfones (IASs) as new HIV-1 NNRTIs. The new IASs 8–37 showed potent inhibition of the HIV-1 WT NL4-3 strain and of the mutant K103N, Y181C, Y188L, and K103N–Y181C HIV-1 strains. Six racemic mixtures, 8, 23–25, 31, and 33, were separated at semipreparative level into their pure enantiomers. The (R)-8 enantiomer bearing the chiral (α-methylbenzyl) was superior to the (S)-counterpart. IAS derivatives bearing the (S) alanine unit, (S)-23, (S,R)-25, (S)-31, and (S)-33, were remarkably more potent than the corresponding (R)-enantiomers. Compound 23 protected hippocampal neuronal cells from the excitotoxic insult, while efavirenz (EFV) did not contrast the neurotoxic effect of glutamate. The present results highlight the chiral IASs as new NNRTIs with improved resistance profile against the mutant HIV-1 strains and reduced neurotoxic effects