Mehran Goulian

A DNA Polymerase-α·Primase Cofactor with Homology to Replication Protein A-32 Regulates DNA Replication in Mammalian Cells

α-Accessory factor (AAF) stimulates the activity of DNA polymerase-α·primase, the only enzyme known to initiate DNA replication in eukaryotic cells ( Goulian, M., Heard, C. J., and Grimm, S. L. (1990) J. Biol. Chem. 265, 13221-13230 ). We purified the AAF heterodimer composed of 44- and 132-kDa subunits from cultured cells and identified full-length cDNA clones using amino acid sequences from internal peptides. AAF-132 demonstrated no homologies to known proteins; AAF-44, however, is evolutionarily related to the 32-kDa subunit of replication protein A (RPA-32) and contains an oligonucleotide/oligosaccharide-binding (OB) fold domain similar to the OB fold domains of RPA involved in single-stranded DNA binding. Epitope-tagged versions of AAF-44 and -132 formed a complex in intact cells, and purified recombinant AAF-44 bound to single-stranded DNA and stimulated DNA primase activity only in the presence of AAF-132. Mutations in conserved residues within the OB fold of AAF-44 reduced DNA binding activity of the AAF-44·AAF-132 complex. Immunofluorescence staining of AAF-44 and AAF-132 in S phase-enriched HeLa cells demonstrated punctate nuclear staining, and AAF co-localized with proliferating cell nuclear antigen, a marker for replication foci containing DNA polymerase-α·primase and RPA. Small interfering RNA-mediated depletion of AAF-44 in tumor cell lines inhibited [methyl-3H]thymidine uptake into DNA but did not affect cell viability. We conclude that AAF shares structural and functional similarities with RPA-32 and regulates DNA replication, consistent with its ability to increase polymerase-α·primase template affinity and stimulate both DNA primase and polymerase-α activities in vitro.

The incorporation of uracil into animal cell DNA in vitro

In the presence of dUTP, net DNA synthesis in vitro is substantially reduced. Small DNA fragments that arise during in vitro DNA synthesis in the presence of dUTP are produced as a result of dUMP incorporation and subsequent post-replication excision repair process initiated by uracil-DNA-glycosylase. The size of the fragments is dependent upon the amount of dUMP incorporated, but unlike the normal 4S intermediates of DNA synthesis, these repair products are not precursors to high molecular weight DNA but are further degraded. The high levels of dUTPase as well as the presence of RNA primers on most nascent DNA pieces (Tseng and Goulian, 1977) suggest that repair of uracil-containing DNA does not contribute to the generation of the small, nascent DNA pieces found during DNA synthesis in this in vitro system.

Ribonucleotides in closed circular mitochondrial DNA from HeLa cells

Abstract Closed circular mitochondrial DNA from HeLa cells is sensitive to both alkali and ribonucleases. The kinetics of ring opening in alkali suggest at least two classes of molecules. One class undergoes rapid breakdown, ultimately to fragments smaller than unit length, in contrast to the second class, which is more resistant to alkaline cleavage and is converted in large part to unit length single strands. Ribonucleases A, T1 and H relax the supercoiled molecules, indicating that the alkali susceptibility is due to the presence of ribonucleotides in the DNA. By comparison with the rate of hydrolysis of RNA, the alkali-resistant class of mitochondrial DNA molecules is estimated to contain approximately 3 ribonucleotides and the alkalisensitive class 10–18.

Mechanism of Thymineless Death

Approximately 30 years ago it was observed by Seymour Cohen that an E. coli mutant that required thymine became non-viable within one generation time after transfer to a medium lacking thymine. This was in contrast to the prolonged tolerance for depletion of precursors for protein or RNA synthesis, or requirements for energy metabolism and carbon sources.

Deoxyuridine triphosphatase: A potential site of interaction with pyrimidine nucleotide analogues

Deoxyuridine triphosphatase has been purified from cultured human lymphoid cells in high yield and stable form by a relatively simple procedure. The properties differed somewhat from those reported previously, e.g. apparent Km, molecular weight, and effects of divalent metals. No other naturally occurring dNTP or NTP serves as substrate, however, the enzyme may be an important site of interaction with intracellular derivatives of analogues of dUrd. It is shown here that deoxyuridine triphosphatase acts on araUTP, 6-azadUTP, 2′-FdUTP, and 2′,3′-dideoxyUTP, but the enzyme has no effect on 5-C1dUTP, 5-BrdUTP, 5-HgdUTP and dUrd-5′[α-thio]triphosphate. For the preparation of one of the analogues, the enzyme, trans-N-deoxyribosylase, from Lactobacillus, was used to prepare the deoxynucleoside from the base, a procedure that may have general usefulness.

A comparison of the enzymatic responses of the DNA polymerases from four RNA tumor viruses

Abstract DNA polymerases from avian, feline, murine and simian RNA tumor viruses exhibit substantial differences in optimal assay conditions and vary widely in their template-primer preferences. Avian DNA polymerase utilizes both natural and synthetic template-primers efficiently in the presence of Mg ++ as well as Mn ++ . By contrast, the mammalian viral DNA polymerases are much more responsive to poly(A)·oligo(dT) than to other template-primers, and exhibit up to 20-fold greater activity with Mn ++ than with Mg ++ . In addition, simian sarcoma virus DNA polymerase shows no detectable response to poly(C)·oligo(dG) over a wide variety of conditions stimulatory to the other viral enzymes.

DNA synthesis in vitro in lysates of Escherichia coli

A system using crude lysates of Escherichia coli PolA- for in vitro synthesis of DNA has properties similar to those described previously for washed DNA - membrane complexes, i.e. short-lived synthesis of DNA, 2-3-fold stimulation by ATP, semiconservative form of synthesis, and most of the product in the size distribution of Okazaki fragments. The proximate substrate is shown to be the deoxynucleoside triphosphate. The lysate system can be fractionated into a particular membrane - DNA complex and a soluble portion, each of which is inactive by itself, but which upon mixing restores activity. The particulate fraction provides template as well as essential protein(s). The soluble fraction contains DNA polymerases II and III both of which are capable of the necessary polymerase function, and a factor that is required for the stimulation of DNA synthesis by ATP. The stimulatory activity can also be assayed by restoration of activity to an aged lysate, which has lost the ATP-dependent synthesis; the activity is not a kinase, and is not an ATP-dependent nuclease, at least of the type currently known. The ATP-dependent stimulatory factor has been partially purified but further purification or characterization of it has been limited by its extreme instability. Both the stimulatory factor and ATP are required for semiconservative synthesis in the lysate, and for synthesis of the short fragments. Similar to results in vivo and in permeable cells the new DNA appears to contain RNA, as judged by the evidence for RNA - DNA junctions from modified nearest neighbor experiments. However, the nucleotides at the RNA - DNA junctions in the lysate system are not specific, in contrast to results in permeabilized Escherichia coli.

On the mechanism for formation of RNA · DNA complexes from lymphocytes

Early intermediates in DNA synthesis by human lymphocytes were studied for the possible association of RNA with nascent DNA. Nucleic acid extracts from cells pulse-labeled with [3H] uridine contain RNA that is associated with DNA in Cs2SO4 equilibrium density gradients. The amount of RNA bound to DNA was greatly reduced by repeated denaturation and equilibrium centrifugation. An apparently similar complex between RNA and DNA was formed in reconstruction experiments in which purified [3H] uridine-labeled RNA was mixed with purified DNA. The association between RNA and DNA could be eliminated in the reconstruction experiments and greatly reduced in extracts from pulse-labeled cells by denaturation and equilibrium centrifugation in the presence of formaldehyde. These studies demonstrate that noncovalent bonding between RNA and DNA can account for most, and possibly all, of the RNA with density close to DNA in Cs2SO4 gradients of nascent DNA preparations. In addition, the results indicate that ribonucleotide, demonstrated by other methods to be covalently bound to nascent DNA, must constitute less than 1/5 of the total nucleotide in the molecule.

Nascent DNA from Phytohemagglutinin-Stimulated Human Lymphocytes

DNA synthesis was studied in human peripheral blood lymphocytes that had been stimulated with phytohemagglutinin. DNA pulse-labeled with [3H]thymidine was fractionated by sucrose gradient centrifugation or by chromatography on hydroxylapatite columns. Nascent DNA was identified as a single-stranded species that sedimented at 4-5S in neutral sucrose gradients and appeared to be precursor to chromosomal DNA in pulse-chase experiments. At least two-thirds of the nascent DNA was released as single strands from high molecular weight DNA without employing a denaturation step. It is concluded that synthesis of DNA by phytohemagglutinin-stimulated lymphocytes involves a low molecular weight, single-stranded, short-lived intermediate similar to that described for other eukaryotic cells.

Three cytoplasmic DNA polymerases that utilize poly(rA)-oligo(dT)

Three DNA polymerases that use poly(rA).oligo(dT) were partially purified from cytoplasmic extracts of cultured mouse cells (after removal of mitochondria), and characterized. One is similar to, and may be the same as, the mitochondrial DNA polymerase gamma. The other two enzymes, one 7.5 S and the other 3.6 S, share some properties with DNA polymerases beta and gamma, e.g. their responses to certain inhibitors; however, they are not clearly identified with any previously well-characterized mammalian DNA polymerases. It is also demonstrated that the response of DNA polymerase gamma to N-ethylmaleimide is template dependent, and that DNA polymerase alpha has an authentic (albeit small) activity with poly(rA).oligo(dT).