HeLa Cell DNA Polymerase Alpha Research Articles

DNA polymerase alpha from HeLa cells synthesizes DNA with high fidelity in a reconstituted replication system

To determine the contribution that DNA polymerase alpha makes to the overall DNA replication fidelity in mammalian systems, we measured the fidelity of replication of the SV40-based shuttle vector, pZ189, in a reconstituted in vitro DNA replication system which contained purified HeLa DNA polymerase alpha (in addition to single-stranded DNA binding protein, topoisomerase II, DNA ligase, 5′ → 3′ exonuclease, ribonuclease H, and SV40 T-antigen). We found that DNA polymerase alpha is highly accurate when carrying out bidirectional replication in this system. This high fidelity of replication by DNA polymerase alpha in the reconstituted replication system contrasts with a relatively low fidelity of gap-filling DNA synthesis on the same target gene by purified HeLa cell DNA polymerase alpha in the absence of other replication factors. The fidelity of DNA replication by DNA polymerase alpha, although relatively high in the reconstituted system, is about 4-fold lower than DNA replication in a crude HeLa cell extract which contains additional replication factors including DNA polymerase delta. These results demonstrate that DNA polymerase alpha has the capacity to replicate DNA with high fidelity when carrying out semiconservative DNA replication in a minimal reconstituted replication system, but additional cellular factors not present in the reconstituted system may contribute to the higher replication fidelity of the crude system.

Murine DNA polymerase alpha fills gaps to completion in a direct assay. Altered kinetics of de novo DNA synthesis at single nucleotide gaps.

DNA polymerase alpha was studied in a direct gap-filling assay. Using a defined template, DNA synthesis was primed from the M13 17-mer universal primer and blocked by an oligonucleotide hybridized 56 nucleotides downstream of the primer. DNA polymerase alpha filled this gap to completion. A time course of the reaction showed that in 50% of the substrate molecules, gaps were filled to completion within 10 min. In another 35% of the molecules the final nucleotide was lacking after 10 min. This nucleotide was added at a reduced rate, and was not incorporated into all of the molecules even after 6 h. The reduced rate of incorporation of the final nucleotide is reflected in an increased Km for de novo incorporation of one nucleotide at a single nucleotide gap (0.7 microM), as opposed to the Km for de novo incorporation of one nucleotide into singly primed M13 DNA (0.18 microM). DNA polymerase alpha purified from murine cells infected with the parvovirus minute virus of mice, and HeLa cell DNA polymerase alpha 2, exhibited the same kinetics of gap filling as did DNA polymerase alpha purified from uninfected Ehrlich ascites murine tumor cells. T4 DNA polymerase filled gaps to completion in this assay. Escherichia coli DNA polymerase I Klenow fragment quantitatively displaced the downstream oligonucleotide, and extended nascent DNA chains for an additional 100 nucleotides. Nicks and single-nucleotide gaps produced in gap-filling reactions by murine DNA polymerase alpha and T4 DNA polymerase were sealed by T4 DNA ligase.

Exonuclease activity associated with a multiprotein form of HeLa cell DNA polymerase alpha. Purification and properties of the exonuclease.

The DNase that is associated with a multiprotein form of HeLa cell DNA polymerase alpha (polymerase alpha 2) has two distinct exonuclease activities: the major activity initiates hydrolysis from the 3' terminus and the other from the 5' terminus of single-stranded DNA. The two exonuclease activities show identical rates of thermal inactivation and coincidental migration during chromatofocusing, glycerol gradient centrifugation, and nondenaturing polyacrylamide gel electrophoresis of the DNase. Moreover, the purified DNase shows a single protein band of Mr 69,000 following nondenaturing polyacrylamide and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 3'----5' exonuclease activity hydrolyzes only single-stranded DNA substrates and the products are 5' mononucleotides. This activity recognizes and excizes mismatched bases at the 3' terminus of double-stranded DNA substrates. The 3'----5' exonuclease does not hydrolyze 3' phosphoryl terminated single-stranded DNA substrates. The 5'----3' exonuclease activity also only hydrolyzes single-stranded DNA substrates. The rate of hydrolysis, however is only about 1/25th the rate of the 3'----5' exonuclease. This exonuclease activity requires a 5' single-stranded terminus in order to initiate hydrolysis and does not proceed into double-stranded regions. The products of hydrolysis by 5'----3' exonuclease are also 5' nucleoside monophosphates.

Resolution of the diadenosine 5',5"'-P1,P4-tetraphosphate binding subunit from a multiprotein form of HeLa cell DNA polymerase alpha.

A diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding subunit has been resolved from a high molecular weight (640,000) multiprotein form of DNA polymerase alpha [deoxynucleoside triphosphate:DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] from HeLa cells [DNA polymerase alpha 2 of Lamothe, P., Baril, B., Chi, A., Lee, L. & Baril, E. (1981) Proc. Natl. Acad. Sci. USA 78, 4723-4727]. The Ap4A binding activity copurifies with the DNA polymerizing activity during the course of purification. Hydrophobic chromatography on butylagarose resolves the Ap4A binding activity from the DNA polymerase. The Ap4A binding activity is protein in nature since the binding of Ap4A is abolished by treatment of the isolated binding activity with proteinase K but is insensitive to treatment with DNase or RNase. The molecular weight of the Ap4A binding protein, as determined by polyacrylamide gel electrophoresis under nondenaturing conditions or by NaDodSO4/polyacrylamide gel electrophoresis after photoaffinity labeling of the protein with [32P]Ap4A is 92,000 or 47,000. The binding activity of this protein is highly specific for Ap4A.

Priming of DNA synthesis by diadenosine 5',5"'-P1,P4-tetraphosphate with a double-stranded octadecamer as a template and DNA polymerase alpha.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) primes DNA synthesis in an in vitro system containing purified HeLa cell DNA polymerase alpha, deoxyadenosine triphosphate, and the double-stranded synthetic octadecamer template 5'-d-(G-G-A-G-G-C-T-T-T-T-T-T-G-G-A-G-G-C) (C-C-T-C-C-G-A-A-A-A-A-A-C-C-T-C-C-G)-d-5'; this octadecamer sequence is part of the origin region of DNA synthesis in simian virus 40. Ap4A is shown to be covalently linked to the first residue of the short deoxynucleotide chain synthesized under these experimental conditions. This template-primer system can initiate the new deoxynucleotide chain but cannot extend it beyond the A . T region.

Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication-specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase III.

The 2-amino substituted derivatives of guanine, N2-(p-n-butylphenyl)guanine (BuPG) and N2-(3',4'-trimethylenephenyl) guanine (TMPG), were synthesized and found to selectively inhibit, respectively, HeLa cell DNA polymerase alpha (po1 alpha) and B. subtilis DNA polymerase III (po1 III). Both purines, like their corresponding uracil analogs, BuAu and TMAU (2,9), were specifically competitive with dGTP in their inhibitory action on their target polymerases. BuPG, the pol alpha-specific purine, was also toxic for HeLa cells in vivo, selectively inhibiting DNA synthesis. These N2-substituted purines, in contrast to the 6-substituted uracils, provide a structural basis for the synthesis of nucleosides and nucleotides with considerable potential as probes for the analysis of the structure of specific replicative DNA polymerases and their function in cellular DNA metabolism.

Association of diadenosine 5‘,5“‘-P1,P4-tetraphosphate binding protein with HeLa cell DNA polymerase alpha.

An electrophoretically homogeneous high molecular weight form (640,000) of HeLa cell DNA polymerase alpha was shown to catalyze DNA synthesis with a variety of di- and oligoriboadenylates and oligodeoxyriboadenylates as primers with poly(dT) as template. Diadenosine 5',5"'-P1,p4-tetraphosphate (Ap4A) can be utilized as a primer with poly(dT) as template and was found to be covalently attached to the 5'-end of the poly(dA) product. An Ap4A binding protein is tightly associated with the high molecular weight form of DNA polymerase alpha. This protein which exhibits high affinity, noncovalent binding of Ap4A is resolved from the multiprotein DNA polymerase alpha complex, along with other accessory proteins, by hydrophobic affinity chromatography on phenyl-Sepharose columns.

HeLa cell DNA polymerase alpha is tightly associated with tryptophanyl-tRNA synthetase and diadenosine 5',5"'-P1,P4-tetraphosphate binding activities.

The purified high molecular weight form of HeLa cell DNA polymerase alpha (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) was shown to associate tightly with several aminoacyl-tRNA synthetase activities. Fractionation of the high molecular weight enzyme on hexylagarose followed by gel filtration, chromatography on phosphocellulose, or polyacrylamide gel electrophoresis under nondenaturing conditions demonstrated copurification of only tryptophanyl-tRNA synthetase [L-tryptophan:tRNATrp ligase (AMP-forming), EC 6.1.1.2] along with DNA polymerase alpha. The high molecular weight (660,000) and low molecular weight (145,000) forms of DNA polymerase alpha were shown to possess a highly specific, noncovalent, diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding activity. The dissociation constants were determined to be 16 and 22 microM, respectively, by utilization of a charcoal adsorption procedure. No high-affinity binding of ATP could be detected. These findings suggest a link between the amino acid activation process and DNA replication in mammalian cells.

Butylanilinouracil: a selective inhibitor of HeLa cell DNA synthesis and HeLa cell DNA polymerase alpha.

A series of 6-anilinouracils, dGTP analogues which selectively inhibit specific bacterial DNA polymerases, were examined for their capacity to inhibit purified DNA polymerases from HeLa cells. The p-n-butyl derivative (BuAU) was found to inhibit DNA polymerase alpha with a Ki of approximately 60 microM. The inhibitory effect of BuAU was reversed specifically by dGTP and was observed only for DNA polymerase alpha; polymerases beta and lambda were not inhibited by drug at concentrations as high as 1 mM. BuAU also was inhibitory in vivo in HeLa cell culture; at 100 microM it reversibly inhibited cell division and selectively depressed DNA synthesis. The results of these studies indicate that BuAU is an inhibitor with considerable potential as a specific probe with which to dissect the structure of mammalian polymerase alpha and its putative role in cellular DNA replication.

Study of DNA synthesis in chromatin isolated from HeLa cells.

When incubated in vitro, HeLa cell chromatin can synthesize DNA at rate comparable to that observed with isolated nuclei. The in vitro DNA synthetic activity of chromatin reflects DNA synthesis in intact cells since chromatin from cells in S phase are several times more active thatn preparations derived from mitotic cells. The requirements for the synthesis of DNA by chromatin preparations are also similar to those of isolated nuclei and the size of the DNA pieces made in both systems is roughly comparable. The chromatin system offers several advantages not available with isolated nuclei. Chromatin will synthesize DNA for a much longer time than isolated nuclei so that larger amounts of DNA can be synthesized in vitro In addition, although chromatin has its own endogenous ability to synthesize DNA, it is markedly stimulated by the presence of exogenously added HeLa cell DNA polymerase alpha, beta, and gamma, and, thus, may provide a new template system for the study of DNA synthesis.

Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid

Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha.

RNA-primed DNA synthesis: specific catalysis by HeLa cell DNA polymerase alpha.

We have analyzed and compared the responses of the three major HeLa cell DNA polymerases (alpha, beta, and gamma) to a HeLa DNA template with short RNA or DNA primers hybridized to it. Only DNA polymerase alpha is able to synthesize DNA covalently bonded to the RNA primer via a 3' yields 5' phosphodiester bond. 32P transfer experiments showed that all combinations of ribo- and deoxyribonucleotides are represented in the RNA-DNA linkages but their distribution is nonrandom. The RNA-DNA linked molecules base-paired to a HeLa DNA template strand represent a possible "natural" in vitro primer-template for DNA polymerases and can be extended by all three DNA polymerases (alpha, beta, and gamma). These findings indicate that DNA polymerases beta and gamma are capable of DNA-primed but not RNA-PRIMED DNA synthesis, while DNA polymerase alpha is capable of both RNA-primed and DAN-primed DNA synthesis.