Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase λ

DNA polymerase λ (pol λ) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol λ either in the absence or in the presence of the human auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A). Our data suggested that (i) pol λ had an intrinsic ability to create mismatches and to incorporate ribonucleotides at nearly physiological Mn++ and Mg++ concentrations; (ii) the sequence of the template-primer could influence the misincorporation frequency of pol λ; (iii) pol λ preferentially generated G:T and G:G mismatches; (iv) RP-A, but not PCNA, selectively prevented misincorporation of an incorrect nucleotide by pol λ, without affecting correct incorporation and (v) this inhibitory effect required a precise ratio between the concentrations of pol λ and RP-A. Possible physiological implications of these findings for the in vivo fidelity of pol λ are discusse

Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase λ

DNA polymerase λ (pol λ) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol λ either in the absence or in the presence of the human auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A). Our data suggested that (i) pol λ had an intrinsic ability to create mismatches and to incorporate ribonucleotides at nearly physiological Mn(++) and Mg(++) concentrations; (ii) the sequence of the template-primer could influence the misincorporation frequency of pol λ; (iii) pol λ preferentially generated G:T and G:G mismatches; (iv) RP-A, but not PCNA, selectively prevented misincorporation of an incorrect nucleotide by pol λ, without affecting correct incorporation and (v) this inhibitory effect required a precise ratio between the concentrations of pol λ and RP-A. Possible physiological implications of these findings for the in vivo fidelity of pol λ are discussed

Human proliferating cell nuclear antigen, poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners.

We addressed the analysis of the physical and functional association of proliferating cell nuclear antigen (PCNA), a protein involved in many DNA transactions, with poly(ADP-ribose) polymerase (PARP-1), an enzyme that plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. We demonstrated that PARP-1 and PCNA co-immunoprecipitated both from the soluble and the DNA-bound fraction isolated from S-phase-synchronized HeLa cells. Immunoprecipitation experiments with purified proteins further confirmed a physical association between PARP-1 and PCNA. To investigate the effect of this association on PARP-1 activity, an assay based on the incorporation of radioactive NAD was performed. Conversely, the effect of PARP-1 on PCNA-dependent DNA synthesis was assessed by a DNA polymerase delta assay. A marked inhibition of both reactions was found. Unexpectedly, PARP-1 activity also decreased in the presence of p21waf1/cip1. By pull-down experiments, we provided the first evidence for an association between PARP-1 and p21, which involves the C-terminal part of p21 protein. This association was further demonstrated to occur also in vivo in MNNG (N-methyl-N'-nitro-N-nitrosoguanidine)-treated human fibroblasts. These observations suggest that PARP-1 and p21 could cooperate in regulating the functions of PCNA during DNA replication/repair

Effect of divalent and monovalent cations on calf thymus PCNA-independent DNA polymerase δ and its 3' → 5' exonuclease

AbstractRecent data suggest that DNA polymerases α and δ might have a coordinate functional role at the replication fork. In this communication we show that Mg2+ is likely the natural metal activator for both enzymes. Mn2+, a known mutagenic agent, is a competitive inhibitor of Mg2+ for DNA polymerase δ and acompetitive for DNA polymerase α. The 3'→ 5' exonuclease activity associated with DNA polymerase δ is not affected upon addition of Mn2+, Be2+, another mutagenic agent, on the other hand, has an inhibitory effect on the 3' → 5' exonuclease, but not on the DNA polymerase δ. The data presented might explain the mutagenic and carcinogenic potential of these two divalent cations

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

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

Molecular basis for the enantioselectivity of HIV-1 reverse transcriptase: Role of the 3′-hydroxyl group of the L-(β)-ribose in chiral discrimination between D- and L-enantiomers of deoxy- and dideoxy-nucleoside triphosphate analogs

In order to identify the basis for the relaxed enantioselectivity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and to evaluate possible cross-resistance patterns between L-nucleoside-, D-nucleoside- and non-nucleoside RT inhibitors, to be utilised in anti-HIV-1 combination therapy, we applied an in vitro approach based on the utilisation of six recombinant HIV-1 RT mutants containing single amino acid substitutions known to confer Nevirapine resistance in treated patients. The mutants were compared on different RNA/DNA and DNA/DNA substrates to the wild type (wt) enzyme for their sensitivity towards inhibition by the D- and L-enantiomers of 2′-deoxy- and 2′,3′-dideoxynucleoside triphosphate analogs. The results showed that the 3′-hydroxyl group of the L-(β)-2′-deoxyribose moiety caused an unfavourable steric hindrance with critic residues in the HIV-1 RT active site and this steric barrier was increased by the Y181I mutation. Elimination of the 3′-hydroxyl group removed this hindrance and significantly improved binding to the HIV-1 RT wt and to the mutants. These results demonstrate the critical role of both the tyrosine 181 of RT and the 3′-position of the sugar ring, in chiral discrimination between D- and L-nucleoside triphosphates. Moreover, they provide an important rationale for the combination of D- and L-(β)-dideoxynucleoside analogs with non-nucleoside RT inhibitors in anti-HIV chemotherapy, since non-nucleoside inhibitors resistance mutations did not confer crossresistance to dideoxynucleoside analog

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

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