The contribution of proofreading to the fidelity ofcatalysis by DNA polymerases has been determined with deoxyribonucleoside [1-thioltriphosphate substrates. These analogues, which contain a sulfur in place of an oxygen on the ar phosphorus, are incorporated into DNA by DNA polymerases at rates similar to those of the corresponding unmodified deoxynucleoside triphosphates. The fidelity of DNA synthesis was measured with *X174 am3 DNA; reversion to wild type occurs most frequently by a single base substitution, a C for a T at position 587. By using avian myeloblastosis virus DNA polymerase and DNA polymerase (3 (enzymes without a proofreading 3'-*5' exonucleolytic activity), substitution ofdeoxycytidine thiotriphosphate in the reaction mixture did not alter fidelity. In contrast, with DNA polymerases from E. coli (DNA polymerase I) and bacteriophage T4 (enzymes containing a proofreading activity), fidelity was markedly reduced with deoxycytidine [1-thio]triphosphate. DNA containing phosphorothioate nucleotides is insensitive to hydrolysis by the exonuclease associated with these prokapyotic DNA polymerases. These combined results indicate that the deoxynucleoside [1-thio]triphosphates have normal base-pairing properties; however, once misinserted by a polymerase, they are not excised by proofreading. Proofreading ofa C:A mismatch at position 587 is thereby found to contribute 20-fold to the fidelity of E. coli DNA polymerase I and a greater amount to the fidelity of bacteriophage T4 DNA polymerase.The ability to correct errors during DNA replication has long been recognized as one important mechanism by which an organism can potentially achieve the highly accurate replication of its genetic information. This concept stems from the observation that prokaryotic DNA polymerases contain an integrally associated 3'--5' exonuclease activity, which can selectively remove mistakes as they occur during polymerization (1). Biochemical support for this concept was obtained by Brutlag and Kornberg (2), who demonstrated that the 3'--5' exonuclease of Escherichia coli DNA polymerase I (Pol I) preferentially removes mismatched bases at primer termini before initiation of polymerization. Support for proofreading in vivo comes from studies with certain mutator (3) and antimutator (4) bacteriophage T4 DNA polymerases. These studies (5-8) correlated spontaneous mutation rates of bacteriophage T4 with the ratio between the polymerization reaction and the excision of a noncomplementary nucleotide at the primer terminus. Also, differences in discrimination between adenosine and its base analogue 2-aminopurine by mutant T4 DNA polymerase (9) can be accounted for by proofreading. Based on the kinetic data with substrate analogues, a number of mathematic models for proofreading have been proposed (10-13).Our continuing interest in determining the relative importance of the several mechanisms available to the cell to achieve high fidelity (14) has led us to assess the contribution of proofreading to accuracy by direct measuremen...

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Hormonal influences on connective tissues have been observed for a variety of hormones and a variety of sites. Gonadal hormones cause accumulation of intercellular ground substance in certain locations; the sex skin of monkeys (1) and the cock's comb (2) are well known examples. The metabolism of sulfated mucopolysaccharide components of connective tissue is known to be influenced by growth hormone (3, 4) and adrenal steroids (5, 6, 7), but only cursory metabolic studies have been reported using sex hormones (8, 9). An attempt has been made to determine whether one gonadal steroid, estradiol, influences the metabolism of connective tissue; and hyaline costal cartilage and aorta were chosen for examination. The incorporation of sulfate labeled with S a5 into these tissues, both in vivo and in vitro, was used as a specific measure of metabolic activity of sulfated mucopolysaccharides. Justification for this view can be found in reports from several laboratories (10-13). Hyaline cartilage was chosen for study because it contains large amounts of chondroitin sulfate and because its response to other hormones is well documented (3-7). Aorta was chosen as an example of arterial connective tissue because the rate of incorporation of sulfate into it is impressive (14) and because some striking sex differences in incidence and severity of arterial disease, both spontaneous and experimentally induced (15-19), have been observed. The metabolism of the mucopolysaccharide components of arteries could conceivably have a bearing upon these differences in incidence of disease. Materials and MethodsAnimals.--All animals used in these experiments were adult rats of the Sprague-Dawley strain initially ranging in weight from 200 to 400 gin., but varying by no more than 20 gin. within any one experiment. These were housed in groups of six and supplied with Purina chow and tap water ad l/b., except where indicated in the text.Hormones and Admlnistratitm.--The following preparations of hormone were used: 1 ml.

The influence of estradiol upon incorporation of isotopes into the sulfated acid mucopolysaccharides of connective tissues of the rat has been studied. Administration of the hormone for 3 weeks significantly reduces the in vitro incorporation of S35-sulfate into cartilage and thoracic aorta without significantly altering the amount of hydrolyzable sulfate in these tissues. The reduction of sulfate incorporation into these tissues by estradiol represents reduced synthesis since the hormone reduces incorporation of sulfate into the acid mucopolysaccharides of the tissue to the same extent and since reduction in incorporation of C14-glucose also parallels S35-sulfate incorporation into acid mucopolysaccharides. The rate of synthesis of sulfated acid mucopolysaccharides is greater in thoracic aorta than abdominal aorta or cartilage. Since the amount of hydrolyzable sulfate is the same in these two portions of aorta, the turnover is also more rapid in the thoracic aorta.

In this paper, we consider the effects of metal ions on the accuracy of catalysis by DNA polymerases. Certain activating and nonactivating metal ions have been shown to diminish the fidelity of DNA synthesis in vitro with a variety of DNA polymerases. There is a significant correlation between the metals that decrease fidelity and those that have been reported to be mutagenic and carcinogenic. Thus, metal carcinogens are no exception to the general postulate that carcinogens can be identified by their interactions with DNA. The Present State of KnowledgeA number of metals have been shown to be mutagens and carcinogens (1) and to affect the accuracy of DNA replication (2). In vivo systems are too complicated to begin to unravel the mechanisms by which metals induce mutations and the effects of metal ions on the fidelity of DNA replication. Our approach to this problem has been to examine DNA synthesis in vitro, to determine the effects of different metal ions on the fidelity of this process, and then to ask whether alterations in the fidelity of DNA synthesis are related to the mutagenic and carcinogenic properties of these metals.Prior to considering these studies in detail, it is instructive to consider the mechanism of DNA synthesis in vitro and methods for measuring fidelity of DNA synthesis.