PubMed Health⌕ Search

Biomedical subjects

F W Perrino

Publications and source records attributed to F W Perrino.

At least 19 recordsLinked to original sources
PubMed1997

Eucaryotic DNA primase does not prefer to synthesize primers at pyrimidine rich DNA sequences when nucleoside triphosphates are present at concentrations found in whole cells.

The critical role of NTP concentration in determining where calf thymus DNA primase synthesizes a primer on a DNA template was examined. Varying the concentration of NTPs dramatically altered the template sequences at which primase synthesized primers. At the low NTP concentrations typically used for in vitro experiments (100 microM), primase greatly preferred to synthesize primers at pyrimidine rich DNA sequences. However, when the concentrations of NTPs were increased to levels typically found in whole cells, primers were now synthesized in all regions of the template. Importantly, synthesis of primers in all regions of the DNA template, not just the pyrimidine rich sequences, is the pattern of primer synthesis observed during DNA replication in whole cells. With low concentrations of NTPs (i.e., Vmax/K(M) conditions), primers are only synthesized at the most preferred synthesis sites, namely, those that are pyrimidine rich. In contrast, under conditions of high NTP concentrations, primer synthesis will occur at the first potential synthesis site to which primase binds. Now, the primase x DNA complex will be immediately converted to a primase x DNA x NTP x NTP complex that is poised for primer synthesis.

Adenosine Triphosphate↗
PubMed1996

Kinetic mechanism of the 3'-->5' proofreading exonuclease of DNA polymerase III. Analysis by steady state and pre-steady state methods.

DNA polymerase III holoenzyme is the major replicative enzyme in Escherichia coli. An important component of the high-fidelity DNA synthesis that is characteristic of DNA polymerase III holoenzyme is the 3'-->5' proofreading exonuclease activity resident in the epsilon subunit. Steady state and pre-steady state conditions have been used to determine equilibrium and Michaelis constants for substrate binding and the rate constant for cleavage by purified epsilon subunit. The steady state kinetic constants are K(m) = 16 +/- 6 microM and kcat = 210 +/- 23 s-1 for degradation of single-stranded DNA by epsilon. These steady state values are in agreement with the rate constants determined for excision of the 3' nucleotide of a dT10 oligomer under pre-steady state conditions. Using a simple two-step model, E + Dn reversible E.Dn-->E + Dn-1, we find K = 12 microM and kf = 280 s-1 for the dT10 substrate. In these experiments, epsilon subunit acts in a distributive manner and product release is not the rate-limiting step. Activity of the epsilon subunit on paired DNA oligonucleotides with zero to three mismatches at the 3' terminus indicates that an additional step is required in the mechanism. In the scheme Dn reversible Dn* + E reversible E.Dn*-->E + Dn-1, the 3' terminus undergoes a conformational change or "melts" before the DNA is a substrate for epsilon subunit. With this additional step, the values for binding of activated substrate and cleavage are the same as those for single-stranded DNA. The kinetics for exonucleolytic degradation of single-stranded, paired, and mispaired oligonucleotides support the model that the rate-limiting step in exonucleolytic proofreading of DNA by epsilon subunit is the DNA-melting step.

Base Composition↗
PubMed1996

Fialuridine and its metabolites inhibit DNA polymerase gamma at sites of multiple adjacent analog incorporation, decrease mtDNA abundance, and cause mitochondrial structural defects in cultured hepatoblasts.

The thymidine analog fialuridine deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) was toxic in trials for chronic hepatitis B infection. One mechanism postulated that defective mtDNA replication was mediated through inhibition of DNA polymerase-gamma (DNA pol-gamma), by FIAU triphosphate (FIALTP) or by triphosphates of FIAU metabolites. Inhibition kinetics and primer-extension analyses determined biochemical mechanisms of FIAU, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) -5-methyluracil (FAU), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil triphosphate (TP) inhibition of DNA pol-gamma. dTMP incorporation by DNA pol-gamma was inhibited competitively by FIAUTP, FMAUTP, and FAUTP (K1=0.015, 0.03, and 1.0 microM, respectively). By using oliginucleotide template-primers. DNA pol-gamma incorporated each analog into DNA opposite a single adenosine efficiently without effects on DNA chain elongation. Incorporation of multiple adjacent analogs at positions of consecutive adenosines dramatically impaired chain elongation by DNA pol-gamma. Effects of FIAU, FMAU, and FAU on HepG2 cell mmtDNA abundance and ultrastructure were determined. After 14 days, mtDNA decreased by 30% with 20 microM FIAU or 20 microM FMAU and decreased less than 10% with 100 microM FAU. FIAU and FMAU disrupted mitochondria and caused accumulation of intracytoplasmic lipid droplets. Biochemical and cell biological findings suggest that FIAU and its metabolites inhibit mtDNA replication, most likely at positions of adenosine tracts, leading to decreased mtDNA and mitochondrial ultrastructural defects.

Antiviral Agents↗
PubMed1995

The effects of cytosine arabinoside on RNA-primed DNA synthesis by DNA polymerase alpha-primase.

Oligonucleotides containing a specific initiation site for polymerase alpha-primase (pol alpha-primase) were used to measure the effects of cytosine arabinoside triphosphate and cytosine arabinoside monophosphate (araCMP) in DNA on RNA-primed DNA synthesis. Primase inserts araCMP at the 3' terminus of a full-length RNA primer with a 400-fold preference over CMP. The araCMP is elongated efficiently by pol alpha in the primase-coupled reaction. Extension from RNA 3'-araCMP is 50-fold less efficient than from CMP, and extension from DNA 3'-araCMP is 1600-fold less efficient than from dCMP. Using araCMP-containing templates, primer synthesis is reduced 2-3-fold, and RNA-primed DNA synthesis is reduced 2-8-fold. The efficiency of polymerization past a template araCMP by pol alpha is reduced 180-fold during insertion of dGMP opposite araCMP and 35-fold during extension from the araCMP:dGMP 3' terminus. These results show that the pol alpha-primase efficiently incorporates araCMP as the border nucleotide between RNA and DNA and suggest that the inhibitory effects of araC most likely result from slowed elongation of pol alpha and less so from inhibition of primer synthesis by primase.

Arabinonucleotides↗
PubMed1995

Initiation of RNA-primed DNA synthesis in vitro by DNA polymerase alpha-primase.

The initiation of new DNA strands at origins of replication in animal cells requires de novo synthesis of RNA primers by primase and subsequent elongation from RNA primers by DNA polymerase alpha. To study the specificity of primer site selection by the DNA polymerase alpha-primase complex (pol alpha-primase), a natural DNA template containing a site for replication initiation was constructed. Two single-stranded DNA (ssDNA) molecules were hybridized to each other generating a duplex DNA molecule with an open helix replication 'bubble' to serve as an initiation zone. Pol alpha-primase recognizes the open helix region and initiates RNA-primed DNA synthesis at four specific sites that are rich in pyrimidine nucleotides. The priming site positioned nearest the ssDNA-dsDNA junction in the replication 'bubble' template is the preferred site for initiation. Using a 40 base oligonucleotide template containing the sequence of the preferred priming site, primase synthesizes RNA primers of 9 and 10 nt in length with the sequence 5'-(G)GAAGAAAGC-3'. These studies demonstrate that pol alpha-primase selects specific nucleotide sequences for RNA primer formation and suggest that the open helix structure of the replication 'bubble' directs pol alpha-primase to initiate RNA primer synthesis near the ssDNA-dsDNA junction.

Animals↗
PubMed1994

Mammalian DNA polymerases alpha, beta, gamma, delta, and epsilon incorporate fialuridine (FIAU) monophosphate into DNA and are inhibited competitively by FIAU Triphosphate.

Fialuridine [FIAU, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5- iodouridine] was used in clinical trials for chronic hepatitis B virus infection and was extremely toxic. Evidence suggested targets of FIAU toxicity included mitochondria, but toxic mechanisms were unclear. Since FIAU is a thymidine analog, we reasoned that triphosphorylated FIAU (FIAUTP) could be incorporated into mitochondrial DNA by DNA pol-gamma and into genomic DNA by DNA polymerases alpha, beta, delta, and epsilon. All five purified mammalian DNA polymerases incorporated FIAUMP into the nascent DNA chain during in vitro DNA synthesis. When FIAUTP was substituted for dTTP, oligonucleotide products were generated efficiently by DNA pol-gamma and were similar to those generated in the presence of the four normal dNTPs. In contrast, oligonucleotide products generated by the four nuclear DNA polymerases in the presence of FIAUTP were significantly reduced in length relative to those generated in the presence of dTTP. In parallel kinetic assays, FIAUTP competitively inhibited the accumulation of radiolabeled dTTP into DNA by DNA pol-gamma. The Ki with DNA pol-gamma was 0.04 microM, the lowest Ki among the mammalian DNA polymerases. Competition between FIAUTP and dTTP and the relative ease of accumulation of FIAUMP in mitochondrial DNA by DNA pol-gamma in vitro together may relate to clinical FIAU toxicity.

Animals↗
PubMed1994

Identification of a 3'-->5'-exonuclease that removes cytosine arabinoside monophosphate from 3' termini of DNA.

Cytosine arabinoside monophosphate (araCMP) at the 3' terminus of DNA constitutes a lesion that impedes further synthesis by DNA polymerase alpha (DNA pol alpha). A biochemical assay has been designed to detect 3'-->5'-exonucleases in cell extracts that remove the 3'-araCMP lesion in an oligonucleotide template-primer and permit subsequent extension by DNA pol alpha. The major 3'-->5'-exonuclease activity in human myeloblast extracts has been purified, and gel filtration chromatography of the purified enzyme indicates that the exonuclease has an apparent native molecular mass of 52 kDa. Incubation of the enzyme with a 5'-32P-labeled araCMP template-primer results in exonucleolytic degradation of the primer exclusively in the 3'-->5' direction, demonstrating that the enzyme is a 3'-->5'-exonuclease. The products of the 3'-->5'-exonuclease reaction are 5'-mononucleotides. The apparent rate of araCMP removal by the exonuclease is approximately the same as the rate of deoxynucleoside monophosphate (dNMP) removal. Furthermore, the apparent rates of 3'-terminal excision are approximately the same whether the oligomer is hybridized to a complementary oligonucleotide, or not, indicating that the enzyme has both single- and double-stranded 3'-->5'-exonuclease activity. The enzyme does not possess 5'-->3'-exonuclease activity, nor is it associated with DNA polymerase activity. In addition, the enzyme does not cleave 3'-phosphoryl-terminated DNA, and it does not cleave RNA. The enzymatic characteristics of the isolated 3'-->5'-exonuclease indicate that it is distinct from previously identified mammalian deoxyribonucleases.

Arabinonucleotides↗
PubMed1993

Proof-reading 3'-->5' exonucleases isolated from rat liver nuclei.

Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of the editing 3'-->5' exonucleolytic activity. Presumably this activity could be effected by the exonucleases non-associated covalently with DNA polymerases. Two 3'-->5' exonucleases of 40 kDa and 50 kDa (exo-40 and exo-5) have been isolated from rat liver nuclei and purified to near homogeneity. They are shown to excise mismatched nucleotides from poly[d(A-T)] template, respectively, 10-fold and 2-fold faster than the matched ones. Upon addition of either of these exonucleases to the DNA polymerase alpha from rat liver or calf thymus, the fidelity of in-vitro reproduction of the primed DNA from bacteriophage phi X174 amber 3 is increased 5-10-fold, levels of exonuclease and DNA-polymerase activities being similar. Extrapolation of in-vitro DNA-replication fidelity to the cellular levels of activities of the exonucleases and the alpha-polymerase suggests that exonucleolytic proof-reading augments the accuracy of DNA synthesis by 2-3 orders of magnitude.

Animals↗
PubMed1993

Primer RNA chain termination induced by 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate. A mechanism of DNA synthesis inhibition.

The studies described herein were aimed at defining the mechanism by which 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (FaraATP), the active intracellular metabolite of fludarabine phosphate, inhibits the synthesis of primer RNA and RNA-primed DNA by the polymerase alpha-primase complex. Incubation of the purified DNA polymerase alpha-primase complex with a poly(dT) template, 500 microM ATP, and increasing concentrations of FaraATP from 2.5 to 50 microM resulted in the progressive accumulation of smaller oligoribonucleotides (2-6 nucleotides) at the expense of the full-length products of DNA primase (7-10 nucleotides). Comparison of the kcat/KM values for incorporation of FaraATP and ATP into oligoribonucleotides revealed that DNA primase incorporated FaraATP 30-fold more efficiently than ATP. FaraAMP was present exclusively at the 3'-termini of the growing primer RNA chains, which prevented further elongation of the primers by DNA primase (primer RNA chain termination). At all FaraATP concentrations tested, inhibition of RNA-primed DNA synthesis was accompanied by primer chain termination. In contrast, DNA polymerase alpha added FaraATP onto full-length primer RNAs about 8-fold less efficiently than dATP, and the incorporation of FaraAMP at the 3'-termini of the primers did not prevent further elongation of these primers by DNA polymerase alpha. These results indicate that primer RNA chain termination is the major effect responsible for the inhibition of RNA-primed DNA synthesis by fludarabine phosphate.

Adenosine Triphosphate↗
PubMed1992

Incorporation of cytosine arabinoside monophosphate into DNA at internucleotide linkages by human DNA polymerase alpha.

The incorporation of cytosine arabinoside monophosphate (araCMP) into DNA at internucleotide linkages by DNA polymerase alpha (DNA pol alpha) has been investigated by using oligonucleotide primed DNA templates. The products of reactions catalyzed by DNA pol alpha in vitro were analyzed on polyacrylamide gels to measure insertion of araCMP, extension from an araCMP 3' terminus, and binding of the enzyme to an araCMP 3' terminus. The results show that insertion of araCMP opposite dGMP in the DNA template is about 3-fold less efficient than insertion of dCMP. Extension from an araCMP 3' terminus by addition of the next complementary nucleotide is approximately 2000-fold less efficient than extension from a correctly base-paired 3' terminus. In the absence of the second substrate, dNTP, DNA pol alpha binds with approximately equal affinities to DNA templates that contain oligonucleotide primers with araCMP or dCMP positioned at the 3' terminus. In the presence of dNTP, the enzyme extends the araCMP 3' terminus or dissociates, but it is not trapped at the araCMP 3' terminus in a nonproductive ternary complex as is observed at the ddCMP 3' terminus. To determine if slow phosphodiester bond formation contributes to the observed extension rate from the araCMP 3' terminus by DNA pol alpha, oligonucleotide primers with araCMP positioned at the 3' terminus were elongated by addition of the alpha-phosphorothioate analogue of the next complementary nucleotide. The rate of extension from araCMP by addition of 2'-deoxyadenosine 5'-O-phosphorothioate (dAMP alpha S) was 6-fold slower than by addition of dAMP, indicating that bond formation is partially rate limiting in the extension reaction. Thus, inefficient extension from the araCMP 3' terminus is the major determinant contributing to the low incorporation frequency of araCMP into DNA by DNA pol alpha, and this inefficiency can be attributed, in part, to slower phosphodiester bond formation at the araCMP 3' terminus.

Adenosine Triphosphate↗
PubMed1991

An EPR study to determine the relative nucleic acid binding affinity of single-stranded DNA-binding protein from Escherichia coli.

A direct quantitative determination by EPR of the nucleic acid binding affinity relationship of the single-stranded DNA-binding protein (SSB) from Escherichia coli at close to physiological NaCl concentration is reported. Titrations of (DUAP, dT)n, an enzymatically spin-labeled (dT)n, with SSB in 20 mM Tris-HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton with either low (5 mM), intermediate (125 mM) or high 200 mM) NaCl content, reveal the formation of a high nucleic acid density complex with a binding stoichiometry (s) of 60 to 75 nucleotides per SSB tetramer. Reverse titrations, achieved by adding (DUAP, dT)n to SSB-containing solutions, form a low nucleic acid density complex with an s = 25 to 35 in the buffer with low NaCl content (5 mM NaCl). The complex with an s = 25 to 35 is converted to the high nucleic acid density complex by increasing the NaCl content to 200 mM. It is, therefore, metastable and forms only under reverse titration conditions in low NaCl. The relative apparent affinity constant Kapp of SSB for various unlabeled single-stranded nucleic acids was determined by EPR competition experiments with spin-labeled nucleic acids as macromolecular probes in the presence of the high nucleic acid density complex. The Kapp of SSB exhibits the greatest affinity for (dT)n as was previously found for T4 gene 32 protein (Bobst, A.M., Langemeier, P.W., Warwick-Koochaki, P.E., Bobst, E.V. and Ireland, J.C. (1982) J. Biol. Chem. 257, 6184) and gene 5 protein (Bobst, A.M., Ireland, J.C. and Bobst, E.V. (1984) J. Biol. Chem. 259, 2130) by EPR competition assays. In contrast, however, SSB does not display several orders of magnitude greater affinity for (dT)n than for other single stranded DNAs as is the case with both gene 5 and T4 gene 32 protein. The relative Kapp values for SSB in the above buffer with 125 mM NaCl are: Kapp(dT)n = 4KappfdDNA = 40Kapp(dA)n = 200Kapp(A)n.

DNA, Bacterial↗
PubMed1990

Hydrolysis of 3'-terminal mispairs in vitro by the 3'----5' exonuclease of DNA polymerase delta permits subsequent extension by DNA polymerase alpha.

Purified DNA polymerase alpha, the major replicating enzyme found in mammalian cells, lacks an associated 3'----5' proofreading exonuclease that, in bacteria, contributes significantly to the accuracy of DNA replication. Calf thymus DNA polymerase alpha cannot remove mispaired 3'-termini, nor can it extend them efficiently. We designed a biochemical assay to search in cell extracts for a putative proofreading exonuclease that might function in concert with DNA polymerase alpha in vivo but dissociates from it during purification. Using this assay, we purified a 3'----5' exonuclease from calf thymus that preferentially hydrolyzes mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by DNA polymerase alpha. This exonuclease copurifies with a DNA polymerase activity that is biochemically distinct from DNA polymerase alpha and exhibits characteristics described for a second replicative DNA polymerase, DNA polymerase delta. In related studies, we showed that the 3'----5' exonuclease of authentic DNA polymerase delta, like the purified exonuclease, removes terminal mispairs, allowing extension by DNA polymerase alpha. These data suggest that a single proofreading exonuclease could be shared by DNA polymerases alpha and delta, functioning at the site of DNA replication in mammalian cells.

Animals↗
PubMed1989

Sequence specificity of pausing by DNA polymerases.

We have constructed recombinant M13 DNA templates containing stretches of oligo (purines) and oligo (pyrimidines). Each of these inserts hinders the advancement of the large fragment of E. coli Pol I during DNA synthesis. The pattern of blockage is independent of changes in KCl or Mg2+ concentrations and pausing is moderately alleviated at lower pH. Blockage is not affected by either the concentration of template or by the position of the DNA primer. The pattern of pause sites is similar for calf thymus DNA polymerase-alpha, implying that replicative barriers are determined by the structure of the DNA at its growing point. There is a lack of correlation between the position of pause sites with different inserts and known alternate DNA structures. Thus, the homo-oligomeric inserts may possess a different structure when complexed with DNA polymerase. This concept accounts for the appearance of unique new upstream and downstream pause sites that result from the insertion of each oligonucleotide.

Base Sequence↗
PubMed1989

Differential extension of 3' mispairs is a major contribution to the high fidelity of calf thymus DNA polymerase-alpha.

The fidelity of DNA polymerase-alpha-primase from calf thymus has been analyzed by measuring mutagenesis in vitro and by site-specific nucleotide misinsertion and mispair extension. Using the phi X174 am3 DNA reversion assay errors are detected at the amber3 site only when both dATP and dCTP are significantly biased during in vitro copying reactions. Analysis of these products on DNA sequencing gels reveals pause sites due to the slow extension of mispaired 3' termini. Measurements of misinsertion rates opposite template A show that the rates of dAMP or dCMP misinsertion are similar and occur 40-50 times more rapidly than dGMP misinsertion. The rate of extension from an A:C mispair is 100- and 400-fold greater than from an A:A mispair and an A:G mispair, respectively. Nucleotide misinsertions to generate all 12 possible mispairs have been measured kinetically on phi X174 DNA templates that contain either A, C, G, or T at position 587. Misinsertion frequencies range from 1/4000 to 1/10(6) depending on the mispairs generated. Extension from all 12 different mispairs was examined by starting with oligonucleotide primers that contain different 3'-terminal mispairs. Rates of extension from mispairs are 10(3) to 10(6) times slower than from correctly paired bases. Extension frequencies were purine:pyrimidine greater than pyrimidine:pyrimidine greater than purine:purine. Lack of extension of misincorporated bases suggests the involvement of exonucleolytic proofreading to enable continued DNA synthesis and to guarantee the high fidelity of eucaryotic DNA replication.

Animals↗
PubMed1989

Extension of mismatched 3' termini of DNA is a major determinant of the infidelity of human immunodeficiency virus type 1 reverse transcriptase.

The unusually high error rate of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) suggests that polymerization errors by this enzyme contribute to the genetic variability of the AIDS virus. We have analyzed the mechanism for HIV-1 RT infidelity by studying two distinct steps that might lead to base substitution mutations: nucleotide misinsertions and elongation from 3'-terminal DNA mispairs. Our results indicate that the capacity of HIV-1 RT to polymerize nucleotides onto mispaired termini is a major factor in the production of mutations by this enzyme. When a noncomplementary dAMP was inserted opposite a template adenine by HIV-1 RT, the nascent 3'-terminal A.A mispair was readily extended by subsequent incorporation of the next complementary nucleotide. The frequencies of nucleotide addition onto 3'-terminal A-A, A-C, and A-G mispairs were determined by quantitating the amount of extended primers with a gel electrophoresis assay and by measuring mutagenesis after hybridization of mismatched primers opposite an amber mutation in bacteriophage phi X174 DNA. The mispair extension frequencies are approximately 50-fold higher by HIV-1 RT than by the mammalian replicative enzyme DNA polymerase alpha.

Base Composition↗
PubMed1989

Proofreading by the epsilon subunit of Escherichia coli DNA polymerase III increases the fidelity of calf thymus DNA polymerase alpha.

Addition of the 3'----5' proofreading exonuclease, epsilon subunit of Escherichia coli DNA polymerase III, to DNA polymerase alpha from calf thymus has been studied. Alone, calf thymus DNA polymerase alpha terminates in vitro DNA synthesis upon insertion of noncomplementary nucleotides. Upon addition of the epsilon subunit, DNA polymerase alpha elongates the newly synthesized DNA as a result of hydrolysis of the 3'-terminal mispair. The fidelity of DNA polymerase alpha in vitro is increased 7-fold by addition of the exonuclease. The functional interaction between DNA polymerase alpha and the epsilon subunit is independent of any detectable physical association. This suggests that a mechanism for proofreading could exist in mammalian cells involving sequential catalysis by DNA polymerase alpha excision of errors by a separate 3'----5' exonuclease, and further elongation onto correctly base-paired 3' termini by DNA polymerase alpha.

Animals↗
PubMed1988

Interaction of a folded chromosome-associated protein with single-stranded DNA-binding protein of Escherichia coli, identified by affinity chromatography.

A single-stranded DNA-binding protein (SSB) affinity column was prepared by optimizing the coupling of Escherichia coli single-stranded DNA-binding protein to Affi-Gel 10. The bound SSB retained its ability to specifically bind single-stranded DNA. When nuclease-treated cell extracts were incubated with the SSB beads overnight at 4 degrees C, a major protein of Mr = 25,000 was bound. At shorter incubation times, two additional proteins of Mr = 32,000 and 36,000 were also detected. In the absence of nuclease treatment, eight additional proteins ranging from Mr = 14,000 to 160,000 also bound to the affinity column. The major Mr = 25,000 protein has been shown to be a folded chromosome-associated protein. Its binding to SSB is strongly enhanced by the addition of DNA polymerase III or DNA polymerase III holoenzyme.

Bacterial Proteins↗