Accumulation of 10-kilobase DNA replication intermediates in cells treated with 3-aminobenzamide.

Abstract

During eukaryotic DNA synthesis there is formation of, in addition to Okazaki fragments, discrete 10-kilobase (kb) DNA replication intermediates. We have investigated the ligation of 10-kb DNA replication intermediates to high molecular weight DNA, using the drug 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase. In human melanoma cells treated with this inhibitor, there is an accumulation of 10-kb DNA. In contrast, in cells treated with aphidicolin, which inhibits DNA polymerase a, there is continued ligation of 10-kb DNA to high molecular weight DNA. Furthermore, using sequential treatment with aphidicolin and 3-aminobenzamide, one can observe the conversion of radiolabeled Okazaki fragments into 10-kb intermediates. The 10-kb DNA pieces are, however, not ligated to high molecular weight DNA in the presence of 3-aminobenzamide. Our results imply that functioning poly(ADP-ribose) synthetase is necessary for the ligation process.Eukaryotic DNA synthesis is very complex and there exist several steps in the synthetic pathway that are not understood (1). For example, we know very little about the steps in the maturation of primary DNA replication intermediates (Okazaki fragments) to high molecular weight DNA. Our inability to examine this process has been due in part to the large size of the eukaryotic genome, which has made it difficult to detect defined DNA replication intermediates that are larger than Okazaki fragments.We have examined the ligation of a large DNA replication intermediate. Using a novel procedure to lyse cells in dilute alkali, we have detected at least two discrete DNA replication intermediates, Okazaki fragments and 10-kilobase (kb) fragments (2-4). The formation of both the Okazaki fragments and 10-kb DNA replication intermediates is dependent on DNA polymerase a (3). However, the ligation of 10-kb DNA to high molecular weight DNA still occurs when DNA polymerase a is inhibited by aphidicolin.Poly(ADP-ribose) synthetase catalyzes the poly(ADP-ribosyl)ation of chromosomal proteins (5)(6)(7)(8). Histones, mainly histone H1, are the major acceptors. Much work has been performed in attempts to understand the biological significance of this secondary modification. It is known that the poly(ADP-ribosyl)ation of histones results in relaxation of polynucleosomes (9, 10) and that the activity of poly(ADPribose) synthetase is dependent on nicks in the DNA (5)(6)(7)(8)11). In cells synchronized with a G1 block, poly(ADP-ribosyl)ation exhibits a cell cycle-dependent oscillation with a maximum coinciding with S phase (12). This observation suggests connection between poly(ADP-ribose) synthetase and S-phase events, such as DNA synthesis.Poly(ADP-ribose) synthetase can be inhibited using the drug 3-aminobenzamide (13), which is a competitive inhibitor that appears to be highly specific. It inhibits the repair of DNA damage induced by various alkylating agents or by yirradiation. Previous reports suggest that it is the ligation of DNA during the repair process that is impaired (14-17). In...

Altered formation of DNA replication intermediates in cells growing in different culture conditions

“…The data are essentially the same as those published earlier (Lonn and Lonn, 1983). Furthermore, earlier experiments have shown that the Okazaki fragments are joined to form 10-kb DNA intermediates, which later give rise to high-molecular-weight DNA (Lonn and Lonn, 1985).…”

“…In different cells different results were obtained on the increase in size of nascent DNA, with a discontinous increase in primary cultures of mouse embryo cells (Kowalski and Cheevers, 1976) and a continuous increase in C3H 10T112 cells (Cordeiro-Stohne and Kaufman, 1985). In some cells long-lived DNA replication intermediates have been detected (Funderud et al, 1978;Lonn and Lonn, 1985), which do not exist in other cells (Cordeiro-Stohne and Kaufman, 1985).…”

Step‐wise progression of mammalian 10‐kb DNA replication intermediates to mature chromatin

“…It is known that alterations of chromatin structure by poly(ADP-ribose) synthetase influence the activity of DNA ligases [7]. In agreement with this we have shown earlier that there is an accumulation of large DNA replication intermediates (10-kb DNA) in human melanoma cells treated with 3-AB [8]. We wished to examine whether this accumulation interferes with the appearance of the post-elongation-stage DNA and the conversion of the new DNA to mature chromatin.…”

Poly(ADP-ribose) polymerase inhibitors suppress UV-induced human immunodeficiency virus type 1 gene expression at the posttranscriptional level.

“…There has been much discussion about the roles played by poly-ADP ribosylation of proteins in various biological functions, including the repair of chemically as well as physically damaged DNA in mammalian cells (4,5,9). Poly-ADP ribosylation has also been implicated in cellular differentiation and replication (10,15,24). Despite numerous reports, mostly concerning biochemical analysis of the reaction with purified poly(ADP-ribose) polymerase, little is known about how poly-ADP ribosylation is involved in these biological functions.…”