Rat DNA polymerase beta (beta-pol) is a 39-kDa protein organized in two tightly folded domains, 8-kDa N-terminal and 31-kDa C-terminal domains, connected by a short protease-sensitive region. The 8-kDa domain contributes template binding to the intact protein, and we now report that the 31-kDa C-terminal domain contributes catalytic activity. Our results show that this domain as a purified proteolytic fragment conducts DNA synthesis under appropriate conditions but the kcat is lower and primer extension properties are different from those of the intact enzyme. A proteolytic truncation of the 31-kDa catalytic domain fragment, to remove a 60-residue segment from the NH2-terminal end, results in nearly complete loss of activity, suggesting the importance of this segment. Overall, these results indicate that the domains of beta-pol have distinct functional roles, template binding and nucleotidyltransferase, respectively; yet, the intact protein is more active for each function than the isolated individual domain fragment.

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Twelve clones of monkey DNA obtained by a procedure that enriches 103-to 104-fold for nascent sequences activated early in S phase (G. Kaufmann, M. Zannis-Hadjopoulos, and R. G. Martin, Mol. Cell. Biol. 5:721-727, 1985) have been examined. Only 2 of the 12 ors sequences (origin-enriched sequences) are unique (orsl and ors8). Three contain the highly reiterated Alu family (ors3, ors9, and orsll). One contains the highly reiterated a-satellite family (orsl2), but none contain the Kpn family. Those remaining contain middle repetitive sequences. Two examples of the same middle repetitive sequence were found (ors2 and ors6). Three of the middle repetitive sequences (the ors2-ors6 pair, orsS, and orslO) are moderately dispersed; one (ors4) is highly dispersed. The last, ors7, has been mapped to the bona fide replication origin of the D loop of mitochondrial DNA. Of the nine ors sequences tested, half possess snapback (intrachain reannealing) properties.Very little is known of the structural features of nuclear mammalian origins of replication. If DNA synthesis is precisely programmed (12,15), then one might expect some common feature(s) of structure or sequence among those origins activated at each point in S phase (3,5,13,29). Indeed, Radford et al. (27) observed an accumulation of repetitive sequences after release from a hydroxyurea block and therefore suggested that origin regions might be enriched for dispersed repetitive sequences. Gronostajski et al. (16a) have purified a cellular protein required for the initiation of adenovirus DNA replication, nuclear factor I, and have shown that HeLa cell sequences can be isolated that preferentially bind to this protein. They have proposed that these sequences might represent HeLa replication origins. On the other hand, it has been observed (41) that short nascent DNA strands are enriched for snapback sequences and has been suggested therefore that origins may be enriched for inverted repeats. Finally, Roth et al. (30) and Montiel et al. (25) have shown that a number of mouse and human DNA sequences, respectively, contain AT-rich regions homologous to the presumptive nuclear replication origins of yeast (ars sequences) and can serve as replication origins for yeast plasmids. They have proposed that these sequences may be mouse and human DNA replication origins.Unlike the situation in mammalian cells, considerable progress toward the characterization of replication origins has been made for Saccharomyces cerevisiae. The ars sequences have been isolated on the basis of their ability to serve as origins for plasmid replication (7,33,34). Curiously, the yeast mitochondrial replication origin also serves as an ars element (18). Both repetitive and unique nuclear ars sequences have been found (8,9,22 tandemly repeated at the telomeres of yeast chromosomes (8,9). The relevance of these findings to mammalian origins is unknown.In mammalian cells, mitochondrial DNA (mtDNA) is known to commence replication at a few closely linked sites with the L strand as the template. Most...

A monoclonal antibody against purified calf DNA polymerase a (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) was used to immunoprecipitate proteins from a crude soluble extract of growing monkey BSC-1 cells. Most attempts to elucidate the subunit structure of DNA polymerase a (a-polymerase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) have relied upon NaDodSO4/polyacrylamide gel electrophoretic analysis of highly purified preparations. However, identification of enzyme subunits by this approach is complicated by the possibilities of both proteolytic degradation and elimination of important enzyme polypeptides during laborious isolation procedures. Clearly, additional methods, not involving laborious purification, are needed for the study of apolymerase polypeptides. Recently, "activity gel" analysis (1-3) of crude homogenates resulted in the identification of a Mr 110,000-120,000 a-polymerase catalytic polypeptide in a number of eukaryotic tissues; in addition, putative a-polymerase catalytic polypeptides also were detected at about Mr 70,000 (1-3). Nevertheless, use of the activity gel method is complicated by the fact that the detection efficiency of apolymerases is low, and some purified a-polymerases are completely unable to produce an activity signal (2). A promising immunological approach, based upon solid-phase immunobinding, was recently reported by Sauer and Lehman (4). Those workers observed a Mr 182,000 polypeptide in crude extracts of Drosophila embryo that cross-reacted with an antiserum raised against the Mr 148,000 polypeptide of purified Drosophila a-polymerase; generally, a-polymerase subunits of Mr > 160,000 had not been reported prior to this work. Lehman and co-workers (5, 6) subsequently showed that the Mr 182,000 polypeptide itself was capable of DNA polymerase catalytic activity.Studies of a-polymerases have been facilitated recently by the development of monoclonal antibodies to these enzymes. Kom and co-workers (7, 8) used a monoclonal antibody to KB cell a-polymerase to localize the enzyme by immunocytofluorescence, and this group and Wahl et al. used a monoclonal antibody to purify the enzyme by immunoaffinity chromatography (9, 10). Masaki et al. (11) found that a monoclonal antibody to calf a-polymerase could distinguish individual species of the enzyme in partially purified preparations from calf thymus, and Matsukage et al. (12) used a monoclonal antibody to chicken a-polymerase to study tissue-specific expression of the enzyme as a function of embryonic development. In the present study, we used a monoclonal antibody approach to elucidate components of mammalian cell lines that share immunological determinants with purified a-polymerase. Proteins in a crude soluble extract from growth-phase monkey cells were subjected to immunoprecipitation with one of our monoclonal antibodies to apolymerase. Immunoprecipitated polypeptides were electrophoresed in NaDodSO4/polyacrylamide gels and then examined for DNA polymerase activ...

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Tightly confluent monkey cell lines BSC-1 and CV-1 held in stale medium for several days exhibited an extremely low level of thymidine incorporation into cellular DNA. Yet, these cells contained a level of alpha-polymerase equal to about 15% of the level in rapidly dividing cells, and they still were capable of supporting replication of SV40 DNA. SV40 infection and culture in stale medium resulted in a four-fold induction of alpha-polymerase in CV-1 cells, whereas no change in alpha-polymerase level was observed in BSC-1 cells. Characterization of alpha-polymerase partially purified from infected CV-1 cells revealed that 80-90% of the enzyme activity was aphidicolin resistant. SV40 DNA replication in resting CV-1 cells, however, was aphidicolin sensitive. SV40 infection of resting CV-1 cells may induce an aphidicolin-resistant enzyme or lead to a modified alpha-polymerase species.