scholarly journals Human DNA polymerase ε is expressed during cell proliferation in a manner characteristic of replicative DNA polymerases

1995 ◽  
Vol 23 (12) ◽  
pp. 2178-2183 ◽  
Author(s):  
Jussi Tuusa ◽  
Lahja Uitto ◽  
Juhani E. Syvaoja
Keyword(s):  
Cell Proliferation ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Human Dna

Detection and characterization of DNA polymerase activity in Toxoplasma gondii

Parasitology ◽  
1993 ◽  
Vol 107 (2) ◽  
pp. 135-139 ◽  
Author(s):  
A. Makioka ◽  
B. Stavros ◽  
J. T. Ellis ◽  
A. M. Johnson
Keyword(s):  
Toxoplasma Gondii ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Sedimentation Coefficient ◽  
Polymerase Activity ◽  
Magnesium Ions ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Approximate Molecular Weight

SUMMARYA DNA polymerase activity has been detected and characterized in crude extracts from tachzoites of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6·4 S, corresponding to an approximate molecular weight of 150000 assuming a globular shape. Like mammalian DNA polymerase α, the DNA polymerase of T. gondii was sensitive to N-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, δ and ε and also cytosine-β-D-arabinofuranoside-5′-triphosphate which is an inhibitor of α polymerase. The activity was inhibited by 2′,3′-dideoxythymidine-5′-triphosphate which is an inhibitor of mammalian DNA polymerase β and γ. Magnesium ions (Mg2+) were absolutely required for activity and its optimal concentration was 6 mM. The optimum potassium (K+) concentration was 50 mM and a higher concentration of K+ markedly inhibited the activity. Activity was optimal at pH 8. Monoclonal antibodies against human DNA polymerase did not bind to DNA polymerase of T. gondii. Thus the T. gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs.

Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle

1988 ◽  
Vol 8 (11) ◽  
pp. 5016-5025
Author(s):  
A F Wahl ◽  
A M Geis ◽  
B H Spain ◽  
S W Wong ◽  
D Korn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Steady State ◽  
Dna Polymerase ◽  
Transformed Cells ◽  
Mrna Levels ◽  
Dna Polymerase Alpha ◽  
Human Dna ◽  
Alpha Gene

We studied the expression of the human DNA polymerase alpha gene during cell proliferation, during cell progression through the cell cycle, and in transformed cells compared with normal cells. During the activation of quiescent cells (G0 phase) to proliferate (G1/S phases), the steady-state mRNA levels, rate of synthesis of nascent polymerase protein, and enzymatic activity in vitro exhibited a substantial and concordant increase prior to the peak of in vivo DNA synthesis. In transformed cells, the respective values were amplified greater than 10-fold. In actively growing cells separated into discrete stages of the cell cycle by counterflow elutriation or by mitotic shakeoff, levels of steady-state transcripts, translation rates, and enzymatic activities of polymerase alpha were constitutively and concordantly expressed at all stages of the cell cycle, with only a moderate elevation prior to the S phase and a slight decline in the G2 phase. These findings support the conclusion that the regulation of human DNA polymerase alpha gene expression is at the transcriptional level and strongly suggest that the regulatory mechanisms that are operative during the entrance of a cell into the mitotic cycle are fundamentally different from those that modulate polymerase alpha expression in continuously cycling cells.

scholarly journals Human DNA Polymerase ι Promotes Replication through a Ring-Closed Minor-Groove Adduct That Adopts a syn Conformation in DNA

2005 ◽  
Vol 25 (19) ◽  
pp. 8748-8754 ◽  
Author(s):  
William T. Wolfle ◽  
Robert E. Johnson ◽  
Irina G. Minko ◽  
R. Stephen Lloyd ◽  
Satya Prakash ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Minor Groove ◽  
Dna Lesions ◽  
Unsaturated Aldehyde ◽  
Cyclic Form ◽  
Human Dna ◽  
A Minor ◽  
Dna Polymerase Ι

ABSTRACT Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from oxidation of polyamines. The reaction of acrolein with the N 2 group of guanine in DNA leads to the formation of a cyclic adduct, γ-hydroxy-1,N 2-propano-2′-deoxyguanosine (γ-HOPdG). Previously, we have shown that proficient replication through the γ-HOPdG adduct can be mediated by the sequential action of human DNA polymerases (Pols) ι and κ, in which Polι incorporates either pyrimidine opposite γ-HOPdG, but Polκ extends only from the cytosine. Since γ-HOPdG can adopt either a ring-closed cyclic form or a ring-opened form in DNA, to better understand the mechanisms that Pols ι and κ employ to promote replication through this lesion, we have examined the ability of these polymerases to replicate through the structural analogs of γ-HOPdG that are permanently either ring closed or ring opened. Our studies with these model adducts show that whereas the ring-opened form of γ-HOPdG is not inhibitory to synthesis by human Pols η, ι, or κ, only Polι is able to incorporate nucleotides opposite the ring-closed form, which is known to adopt a syn conformation in DNA. From these studies, we infer that (i) Pols η, ι, and κ have the ability to proficiently replicate through minor-groove DNA lesions that do not perturb the Watson-Crick hydrogen bonding of the template base with the incoming nucleotide, and (ii) Polι can accommodate a minor-groove-adducted template purine which adopts a syn conformation in DNA and forms a Hoogsteen base pair with the incoming nucleotide.

Incorporation of Selected Nucleoside Phosphonates and Anti-Human Immunodeficiency Virus Nucleotide Analogues into DNA by Human DNA Polymerases α, β and γ

1997 ◽  
Vol 8 (3) ◽  
pp. 187-195 ◽  
Author(s):  
T Cihlar ◽  
MS Chen
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Immunodeficiency Virus ◽  
Deoxynucleoside Triphosphate ◽  
Template Dna ◽  
Dna Polymerase Γ ◽  
Incorporation Efficiency

Incorporation of selected diphosphates of nucleoside phosphonates and triphosphates of currently approved anti-human immunodeficiency virus nucleoside analogues into DNA by human DNA polymerases α, β and γ was studied. All three polymerases were able to incorporate diphosphates of 9-(2-phosphonomethoxyethyl)adenine (PMEApp), 9-(2-phosphonomethoxyethyl)guanine (PMEGpp), ( R)-9-(2-phosphonomethoxypropyl)adenine (PMPApp), ( R)-9-(2-phosphononomethoxypropyl)-2,6-diaminopurine (PMPDAPpp) and ( 2R,5R)-9-[2,5-dihydro-5-(phosphonomethoxy)-2-furanyl]adenine (D4APpp) into primer/template DNA of defined sequence. After incorporation, these nucleoside phosphonates acted as terminators of primer extension. Kinetic constants of their incorporation were determined and compared with those for incorporation of ddATP, ddCTP, (-)-2′-deoxy-3′-thiacytidine triphosphate (3TC-TP), 2′,3′-didehydro-3′-deoxythymidine triphosphate (d4T-TP) and 3′-azido-3′-deoxythymidine triphosphate (AZT-TP). Relative efficiencies of incorporation (percentage of the incorporation efficiency for the corresponding natural deoxynucleoside triphosphate) by DNA polymerase a ranged from 0.05% for 3TC-TP to 51% for PMEGpp. DNA polymerase β catalysed the incorporation with relative efficiencies ranging from 0.014% for AZT-TP to 125% for ddCTP, and efficiencies of incorporation by DNA polymerase γ varied between 0.13% for 3TC-TP and 25% for ddCTP. Generally, the lowest incorporation efficiencies with all three polymerases were found for PMPApp (0.06–1.4%) and PMPDAPpp (0.075–2.2%).

scholarly journals Kinetic Interaction of the Diphosphates of 9-(2-phosphonylmethoxyethyl)adenine and Other anti-HIV Active Purine Congeners with HIV Reverse Transcriptase and Human DNA Polymerases α, β and γ

1995 ◽  
Vol 6 (4) ◽  
pp. 217-221 ◽  
Author(s):  
J. M. Cherrington ◽  
S. J. W. Allen ◽  
N. Bischofberger ◽  
M. S. Chen
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Inhibitory Effects ◽  
Hiv Reverse Transcriptase ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Dna Template ◽  
Anti Hiv ◽  
Micromolar Range

The inhibitory effects of the diphosphates of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and its analogues on HIV reverse transcriptase and human DNA polymerases α, β, and γ have been studied. The analogues investigated are the diphosphates of 9-(2-phosphonylmethoxypropyl)adenine (PMPApp), 9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine (PMPDAPpp), and (2R,5R)-9-[2,5-dihydro-5-(phosphonyl methoxy)-2-furanyl]adenine (D4APpp). These four compounds are much more inhibitory to HIV reverse transcriptase when an RNA template rather than a DNA template is used. The Ki, values for the four compounds range from 11 to 22 nM with an RNA template. The Ki, values for ddCTP and AZTTP are 54 nM and 8 nM, respectively. PMEApp and its analogues show varying degrees of inhibition of the human DNA polymerases. The Ki, values for PMEApp, PMPApp and PMPDAPpp against DNA polymerase α are in the micromolar range, while D4APpp is a poor inhibitor of this enzyme with a Ki, value of 65.9 μM. The inhibition of DNA polymerase β by PMEApp, PMPApp and D4APpp is minimal, while PMPDAPpp shows higher inhibition of DNA polymerase β with a Ki, value of 9.71 μM. The Ki, values for PMEApp and D4APpp against DNA polymerase γ are submicromolar, while PMPApp and PMPDAPpp are much less inhibitory to this enzyme. For comparison, ddCTP was found to be a more potent inhibitor of DNA polymerases β and γ than the diphosphates of PMEA and its analogues.

scholarly journals Replication pastO6-Methylguanine by Yeast and Human DNA Polymerase η

2000 ◽  
Vol 20 (21) ◽  
pp. 8001-8007 ◽  
Author(s):  
Lajos Haracska ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Brain Tumors ◽  
Dna Polymerase ◽  
Double Mutant ◽  
Malignant Tumors ◽  
Alkylating Agents ◽  
Dna Polymerases ◽  
Clinical Effectiveness ◽  
Induced Mutations ◽  
Human Dna ◽  
Biochemical Studies

ABSTRACT O 6-Methylguanine (m6G) is formed by the action of alkylating agents such asN-methyl-N′-nitro-N-nitrosoguanidine (MNNG) on DNA. m6G is a highly mutagenic and carcinogenic lesion, and it presents a block to synthesis by DNA polymerases. Here, we provide genetic and biochemical evidence for the involvement of yeast and human DNA polymerase η (Polη) in the replicative bypass of m6G lesions in DNA. The formation of MNNG-induced mutations is almost abolished in therad30Δ pol32Δ double mutant of yeast, which lacks theRAD30 gene that encodes Polη and the Pol32 subunit of DNA polymerase δ (Polδ). Although Polδ can function in the mutagenic bypass of m6G lesions, our biochemical studies indicate that Polη is much more efficient in replicating through m6G than Polδ. Both Polη and Polδ insert a C or a T residue opposite from m6G; Polη, however, is more accurate, as it inserts a C about twice as frequently as Polδ. Alkylating agents are used in the treatment of malignant tumors, including lymphomas, brain tumors, melanomas, and gastrointestinal carcinomas, and the clinical effectiveness of these agents derives at least in part from their ability to form m6G in DNA. Inactivation of Polη could afford a useful strategy for enhancing the effectiveness of these agents in cancer chemotherapy.

scholarly journals Mutagenesis mechanism of the major oxidative adenine lesion 7,8-dihydro-8-oxoadenine

2020 ◽  
Vol 48 (9) ◽  
pp. 5119-5134 ◽  
Author(s):  
Myong-Chul Koag ◽  
Hunmin Jung ◽  
Seongmin Lee
Keyword(s):  
Reactive Oxygen Species ◽  
Crystal Structures ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Reactive Oxygen ◽  
Minor Groove ◽  
Oxygen Species ◽  
Base Pairing ◽  
Human Dna ◽  
Hoogsteen Base Pairing

Abstract Reactive oxygen species generate the genotoxic 8-oxoguanine (oxoG) and 8-oxoadenine (oxoA) as major oxidative lesions. The mutagenicity of oxoG is attributed to the lesion's ability to evade the geometric discrimination of DNA polymerases by adopting Hoogsteen base pairing with adenine in a Watson–Crick-like geometry. Compared with oxoG, the mutagenesis mechanism of oxoA, which preferentially induces A-to-C mutations, is poorly understood. In the absence of protein contacts, oxoA:G forms a wobble conformation, the formation of which is suppressed in the catalytic site of most DNA polymerases. Interestingly, human DNA polymerase η (polη) proficiently incorporates dGTP opposite oxoA, suggesting the nascent oxoA:dGTP overcomes the geometric discrimination of polη. To gain insights into oxoA-mediated mutagenesis, we determined crystal structures of polη bypassing oxoA. When paired with dGTP, oxoA adopted a syn-conformation and formed Hoogsteen pairing while in a wobble geometry, which was stabilized by Gln38-mediated minor groove contacts to oxoA:dGTP. Gln38Ala mutation reduced misinsertion efficiency ∼55-fold, indicating oxoA:dGTP misincorporation was promoted by minor groove interactions. Also, the efficiency of oxoA:dGTP insertion by the X-family polβ decreased ∼380-fold when Asn279-mediated minor groove contact to dGTP was abolished. Overall, these results suggest that, unlike oxoG, oxoA-mediated mutagenesis is greatly induced by minor groove interactions.

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