Okazaki fragment processing: modulation of the strand displacement activity of DNA polymerase delta by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1.

DNA polymerase (pol) delta is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol delta has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA/DNA segment of the preexisting downstream fragment generating an intermediate flap structure that is the target for the Dna2 and flap endonuclease-1 (Fen 1) endonucleases. Using a single-stranded minicircular template with an annealed RNA/DNA primer, we could measure strand displacement by pol delta coupled to DNA synthesis. Our results suggested that pol delta alone can displace up to 72 nucleotides while synthesizing through a double-stranded DNA region in a distributive manner. Proliferating cell nuclear antigen (PCNA) reduced the template dissociation rate of pol delta, thus increasing the processivity of both synthesis and strand displacement, whereas replication protein A (RP-A) limited the size of the displaced fragment down to 20-30 nucleotides, by generating a "locked" flap DNA structure, which was a substrate for processing of the displaced fragment by Fen 1 into a ligatable product. Our data support a model for Okazaki fragment processing where the strand displacement activity of DNA polymerase delta is modulated by the concerted action of PCNA, RP-A and Fen 1.

Different effects of methotrexate on DNA mismatch repair proficient and deficient cells.

Antifolates exert their antiproliferative activity through the inhibition of dihydrofolate reductase and, as a consequence, of thymidylate synthesis, thereby inducing nucleotide misincorporation and impairment of DNA synthesis. We investigated the processes involved in the repair of antifolate-induced damage and their relationship with cell death. Since misincorporated bases may be removed by DNA mismatch repair (MMR), the study was carried out on the MMR-proficient human cell lines HeLa and HCT116+chr3, and, in parallel, on the MMR-deficient cell lines HeLa cell-clone12, defective in the protein hPMS2, and HCT116, with an inactive hMLH1. After treatment with methotrexate (MTX), we observed that DNA repair synthesis occurs independently of the cellular MMR function. Clear signs of apoptosis such as nuclear shrinkage, chromatin condensation and degradation, DNA laddering, and poly (ADP-ribose) polymerase (PARP) proteolysis, were visible in both MMR(+) and MMR(-) cells. Remarkably, cell viability was lower and the apoptotic process was triggered more efficiently in the MMR-competent cells.

Replication protein A as a "fidelity clamp" for DNA polymerase alpha.

The current view of DNA replication in eukaryotes predicts that DNA polymerase alpha (pol alpha)-primase synthesizes the first 10-ribonucleotide-long RNA primer on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. Subsequently, pol alpha elongates such an RNA primer by incorporating about 20 deoxynucleotides. pol alpha displays a low processivity and, because of the lack of an intrinsic or associated 3'--> 5' exonuclease activity, it is more error-prone than other replicative pols. Synthesis of the RNA/DNA primer catalyzed by pol alpha-primase is a critical step in the initiation of DNA synthesis, but little is known about the role of the DNA replication accessory proteins in its regulation. In this paper we provide evidences that the single-stranded DNA-binding protein, replication protein A (RP-A), acts as an auxiliary factor for pol alpha playing a dual role: (i) it stabilizes the pol alpha/primer complex, thus acting as a pol clamp; and (ii) it significantly reduces the misincorporation efficiency by pol alpha. Based on these results, we propose a hypothetical model in which RP-A is involved in the regulation of the early events of DNA synthesis by acting as a "fidelity clamp" for pol alpha.

The vestigial gene of Drosophila melanogaster is involved in the formation of the peripheral nervous system: genetic interactions with the scute gene.

The vestigial (vg) gene of Drosophila melanogaster encodes a novel nuclear protein which is required for wing formation but its function is not restricted to this developmental process. In this report, we have examined the possible role of vestigial in the formation of the peripheral nervous system. We found that most vestigial mutants display ectopic or missing macrochaetae. We also show that vestigial interacts in a dose dependant manner with different alleles of the scute gene and with the extramacrochaetae gene and that mis-expression of the vestigial gene in the wing disc increases the domain of scute expression and allows the formation of supernumerary ectopic bristles. From these results, we conclude that vestigial affects scute expression and plays a role in the differentiation of the peripheral nervous system.

Poly(ADP-ribose) synthesis: a useful parameter for identifying apoptotic cells.

Cell death by apoptosis was analysed in HeLa cells either treated with the antitumoral drug bleomycin or depleted of growth factors by long-term culture without medium change. The interference of apoptosis with normal cell cycle progression was followed by flow cytometry in cells stained with propidium iodide and with antibody to S-phase-related PCNA protein. Bleomycin-treated cells showed a net accumulation in G2/M phase paralleled by the appearance of material with a subdiploid DNA content. Cells with a subdiploid DNA content were also present in growth factor-depleted cultures and were shown to derive from all the cell cycle phases. To identify apoptotic features in HeLa cell cultures, we applied a recently developed assay based on the simultaneous analysis in the single cell of three parameters, namely chromatin condensation, DNA degradation and poly(ADP-ribose) synthesis. Apoptotic cells were visualized by sequential reactions: Hoechst staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling assay and immunoreaction with anti-poly(ADP-ribose) monoclonal antibody. Positive reactions were obtained for cells at different stages of the apoptotic programme showing condensed nuclei, fragmented chromatin and apoptotic bodies.

Vestigial gene expression in Drosophila melanogaster is modulated by the dTMP pool.

The vestigial (vg) gene of Drosophila melanogaster encodes a nuclear protein which plays a key role in wing formation but is also involved in other developmental processes. We have previously shown that depletion of the dTMP pool by aminopterin, an inhibitor of the enzyme dihydrofolate reductase, or by fluorodeoxyuridine, an inhibitor of thymidylate synthetase, induces nicks in the wings of wild-type flies and a strong vg phenotype in vgBG/+ flies and also in individuals heterozygous for a deficiency of the vg locus (vgB/+). Furthermore, specific alterations of the vg locus, caused by intronic insertions, are associated with resistance to these drugs. In this paper, we show that: (1) depletion of the dTMP pool by aminopterin leads to a decrease in the amount of vg transcripts; (2) insertion of the retrotransposon 412 in the vgBG mutant, which is resistant to aminopterin, leads to the formation of a truncated transcript that is prematurely terminated in the long terminal repeat of this transposable element; and (3) aminopterin also affects the level of this truncated transcript. These results indicate that alterations of the wing by inhibitors of dTMP synthesis are caused by an effect of these drugs on levels of vg transcripts; the resistance to such agents observed for the vgBG strain is not due to a qualitatively different effect of this drug on the vg transcript but, rather, is related to the expression of a modified Vg protein encoded by a truncated transcript. These results are compatible with a role for vestigial in modulating cell proliferation.