Calf thymus DNA polymerases .alpha. and .delta. are capable of highly processive DNA synthesis

Sabatino, Ralph D.; Myers, Thomas W.; Bambara, Robert A.; Kwon-Shin, O.; Marraccino, Robert L.; Frickey, Paul H.

doi:10.1021/bi00408a050

Cited by 39 publications

(13 citation statements)

References 36 publications

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“…Under these conditions most primers initiated no synthesis at all, whereas DNA synthesis starting from the few primers that were bound was the result of only one stretch of processive activity. The length of extension of the utilized 3Ј-OH primers reflects the processivity of the enzymes (50). Oligo(dT) [12][13][14][15][16][17][18] was hybridized to poly(dA) 200 at a weight ratio of 1:10, the equivalent of a molar ratio of 1.9:1.…”

Section: Resultsmentioning

confidence: 99%

DNA Ligase I Selectively Affects DNA Synthesis by DNA Polymerases δ and ε Suggesting Differential Functions in DNA Replication and Repair

Mossi

Ferrari

Hübscher

1998

Journal of Biological Chemistry

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The joining of single-stranded breaks in doublestranded DNA is an essential step in many important processes such as DNA replication, DNA repair, and genetic recombination. Several data implicate a role for DNA ligase I in DNA replication, probably coordinated by the action of other enzymes and proteins. Since both DNA polymerases ␦ and ⑀ show multiple functions in different DNA transactions, we investigated the effect of DNA ligase I on various DNA synthesis events catalyzed by these two essential DNA polymerases. DNA ligase I inhibited replication factor C-independent DNA synthesis by polymerase ␦. Our results suggest that the inhibition may be due to DNA ligase I interaction with proliferating cell nuclear antigen (PCNA) and not to a direct interaction with the DNA polymerase ␦ itself. Strand displacement activity by DNA polymerase ␦ was also affected by DNA ligase I. The DNA polymerase ␦ holoenzyme (composed of DNA polymerase ␦, PCNA, and replication factor C) was inhibited in the same way as the DNA polymerase ␦ core, strengthening the hypothesis of a PCNA interaction. Contrary to DNA polymerase ␦, DNA synthesis by DNA polymerase ⑀ was stimulated by DNA ligase I in a PCNA-dependent manner. We conclude that DNA ligase I displays different influences on the two multipotent DNA polymerases ␦ and ⑀ through PCNA. This might be of importance in the selective involvement in DNA transactions such as DNA replication and various mechanisms of DNA repair.DNA ligases play essential roles in important cellular pathways, such as DNA replication, DNA recombination, and DNA repair, by joining single-and double-stranded breaks in an ATP-dependent manner (1). Four DNA ligases (I, II, III, and IV), the functions of which are not yet completely understood, have been identified in mammalian cells (2). Human DNA ligase I is a monomer of 102 kDa (3) composed of two clearly distinct regions as follows: a highly conserved 78-kDa C-terminal domain containing the active site (4), and a 24-kDa Nterminal region that is not required for ligase activity but contains the nuclear localization signal and directs the enzyme to sites of DNA replication (5). Several of the following observations indicate an involvement of DNA ligase I in DNA replication: (i) DNA ligase I is responsible for a major part of DNA ligase activity in proliferating mammalian cells (6 -9); (ii) cytostaining experiments with antibodies against DNA ligase I showed that the enzyme co-localizes in the nucleus with DNA polymerase (pol) 1 ␣ (10); (iii) the enzyme co-purifies with a protein complex competent in in vitro SV40 DNA replication (11); (iv) a mutation in the DNA ligase I gene in the human 46BR cell line leads to a delay in the joining of the Okazaki fragments (12). These, together with several other observations (3, 13), imply an important role of DNA ligase I in DNA replication as well as in DNA repair (5,14). Experiments by Mackenney et al. (15) suggest that DNA ligase I, through its Nterminal region, interacts with other proteins.The most important proteins in...

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Section: Resultsmentioning

confidence: 99%

DNA Ligase I Selectively Affects DNA Synthesis by DNA Polymerases δ and ε Suggesting Differential Functions in DNA Replication and Repair

Mossi

Ferrari

Hübscher

1998

Journal of Biological Chemistry

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“…The synthetic activity of some of these forms is not stimulated by PCNA (22,24), and only one form of DNA polymerase 8 isolated from fetal bovine thymus and the enzyme isolated from human placenta require PCNA for efficient synthesis on the model template-primer poly(dA)-oligo(dT) (8,21). The form of DNA polymerase 8 from calf thymus that interacts with PCNA has a subunit structurei.e., two subunits of M, 125,000 and 48,000 (14), very similar to that of yeast DNA polymerase III, which consists of two subunits of Mr 125,000 and 55,000 (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…LA). The other forms of calf thymus DNA polymerase 8 with different, more complex, subunit structures, are unaffected by PCNA (22,24). DNA Strain BJ405 was grown with aeration in 3 liters ofYPD medium at 300C to OD6w = 1.0.…”

Section: Discussionmentioning

confidence: 99%

The yeast analog of mammalian cyclin/proliferating-cell nuclear antigen interacts with mammalian DNA polymerase delta.

Bauer

Burgers

1988

Proc. Natl. Acad. Sci. U.S.A.

137

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DNA polymerase m from Saccharomyces cerevisiae is analogous to the mammalian DNA polymerase 8 by several criteria, including an increased synthetic activity on poly(dA)oligo(dT) (40:1 nucleotide ratio) in the presence ofcalf thymus proliferating-cell nuclear antigen (PCNA), or cyclin.This stimulation assay has been used to purify the yeast analog of PCNA/cyclin (yPCNA) to homogeneity. yPCNA is a trimer or tetramer (Mr 82,000) of identical subunits with a denatured Mr of 26,000. On a molar basis yPCNA and calf thymus PCNA/cyclin are equally active in stimulating DNA synthesis by DNA polymerase Im. About 10 times more yPCNA than calf thymus PCNA/cyclin is needed, however, to stimulate calf thymus DNA polymerase 8, and the degree of stimulation obtained at saturating levels of yPCNA is a factor of 2-3 less than with calf thymus PCNA/cyclin. Both stimulatory proteins exert their effect in an identical fashion, i.e., by increasing the processivity of the DNA polymerase. Yeast DNA polymerases I and H and calfthymus DNA polymerase a are not stimulated by yPCNA. Treatment of logarithmic-phase cells with hydroxyurea blocks them in the S phase and produces a 4-to 5-fold increase in yPCNA.Proliferating-cell nuclear antigen (PCNA), also known as cyclin, is a cell cycle-regulated protein present at high levels in rapidly dividing cells (1-4). The intranuclear localization of PCNA/cyclin to regions of active DNA synthesis indicates that PCNA/cyclin plays a role in DNA replication (5). Recently, it has been shown that PCNA is required for in vitro simian virus 40 (SV40) DNA replication (6,7). In its absence, early replicative intermediates accumulate and leadingstrand DNA synthesis is impaired (7).The additional identification of PCNA/cyclin as an accessory factor of DNA polymerase 8 (8-10) suggests, in conjunction with the SV40 DNA replication data, a role for a complex of DNA polymerase 8 and PCNA/cyclin in processive leading-strand DNA synthesis. We have recently isolated from the yeast Saccharomyces cerevisiae a third nuclear DNA polymerase, which we call DNA polymerase III and which shows properties closely analogous to the mammalian DNA polymerase 8 (11). These include a proofreading 3' 5' exonuclease activity, sensitivity to the drug aphidicolin, and relative resistance to the nucleotide analog N2-[p-(n-butyl)phenylJdGTP (12). In addition, calf thymus PCNA/cyclin interacts with DNA polymerase III and promotes processive DNA synthesis by the enzyme (13). This finding prompted us to identify and purify a PCNA/cyclin-like activity in yeast by using stimulation of DNA polymerase III activity as an assay. The purification of this activity and its interaction with DNA polymerase III, as well as calf thymus DNA polymerase 8, is presented in this paper. Because the name cyclin implies that the expression of the gene and/or the activity of the protein is restricted to a part of the cell cycle, we prefer to use the more neutral term yPCNA (yeast proliferating-cell nuclear antigen) for the activity in yeast, in the absence...

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“…At some point early in replication, RF-C and PCNA displace polymerase a at the 3' terminus of the leading strand. This might easily occur due to the limited processivity of polymerase a under these conditions (27). We propose that a similar event occurs on the lagging strand, in which DNA polymerase a-primase, after synthesizing a short stretch of DNA, is displaced by RF-C and PCNA at the 3' terminus.…”

Section: Interaction Of Isolatedmentioning

confidence: 90%

A 5' to 3' exonuclease functionally interacts with calf DNA polymerase epsilon.

Siegal

Turchi

Myers

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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Analysis of fractions containing purified DNA polymerase e from calf thymus has revealed the presence of a 5' to 3' exonuclease activity that is specific for a single strand of duplex DNA. This activity is capable of degrading a 3'-labeled oligonucleotide hybridized to M13mpl8 DNA. When a second oligonucleotide primer is anneal 3 bases upstream, degradation of the downstream primer is strictly dependent on DNA synthesis from the upstream primer. Replacement We have recently demonstrated the presence of two forms of DNA polymerase E in calf thymus extracts, designated E and E* (1). Both forms possess intrinsic DNA polymerase and single strand-specific 3' to 5' exonuclease activities. DNA polymerases E and 8* have molecular masses of 210 and 145 kDa, respectively. DNA polymerase E* can be proteolytically derived from DNA polymerase 8 during purification. We considered that this situation might be analogous to DNA polymerase I and the Klenow fragment from Escherichia coli (2, 3). In this case, limited proteolysis of DNA polymerase I results in removal of a domain containing the 5' to 3' exonuclease. The remaining DNA polymerase retains the capacity to exonucleolytically degrade single-stranded DNA in the 3' to 5' direction. However, we (4) and others (5) have examined the single-stranded exonuclease activity of DNA polymerase E from different sources and found it to demonstrate exclusively 3' to 5' directionality.Recent work on the 5' to 3' exonuclease of the DNA polymerase from Thermus aquaticus indicated that this activity is specific for one strand of double-stranded DNA (6, 7). Utilizing a template similar to that described by Longley et al. (6), we have detected a 5' to 3' exonucleolytic activity in fractions of DNA polymerase E. This activity has been purified to near homogeneity and its molecular mass has been determined to be 56 kDa as judged by SDS/PAGE. Thus, it appears that this activity represents a polypeptide other than polymerase E. Based on enzymatic and physical properties of the exonuclease, it may be a homolog of similar enzymes found in mouse cells (8) and HeLa cells (9). If so, in addition to the enzymologic similarity with the proteins described in these two reports, we find that the exonuclease specifically interacts with homologous DNA polymerase E, and not polymerases a and 8, in a two-primer assay described here. Based on these data, we propose a role for the complex of exonuclease and polymerase in DNA metabolism. MATERIALS AND METHODSMaterials. Synthetic oligodeoxyribonucleotides were from Genosys (The Woodlands, TX) and were purified by PAGE. The synthetic oligoribonucleotide corresponding to the -20 universal sequencing primer (5'-GUAAAACGACGGC-CAGU-3') was from National Bioscience Incorporated (Minneapolis). All nucleotides were Ultrapure from Pharmacia. The -20 universal sequencing primer (20 pmol) was 5' phosphorylated with T4 polynucleotide kinase (United States Biochemical) and 1 mM ATP in the buffer supplied by the manufacturer. The exonuclease substrate was created by...

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Calf thymus DNA polymerases .alpha. and .delta. are capable of highly processive DNA synthesis

Cited by 39 publications

References 36 publications

DNA Ligase I Selectively Affects DNA Synthesis by DNA Polymerases δ and ε Suggesting Differential Functions in DNA Replication and Repair

DNA Ligase I Selectively Affects DNA Synthesis by DNA Polymerases δ and ε Suggesting Differential Functions in DNA Replication and Repair

The yeast analog of mammalian cyclin/proliferating-cell nuclear antigen interacts with mammalian DNA polymerase delta.

A 5' to 3' exonuclease functionally interacts with calf DNA polymerase epsilon.

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