THE EFFECTS OF AGE AND OESTRONE TREATMENT ON DNA POLYMERASE ACTIVITY IN ANTERIOR PITUITARY GLANDS OF MALE RATS

1973 ◽  
Vol 59 (1) ◽  
pp. 107-119 ◽  
Author(s):  
ANDREA MASTRO ◽  
W. C. HYMER
Keyword(s):  
Dna Polymerase ◽  
Anterior Pituitary ◽  
Reaction Medium ◽  
Polymerase Activity ◽  
Male Rats ◽  
Synthetic Activity ◽  
Cytoplasmic Fraction ◽  
Isolated Nuclei ◽  
Pituitary Glands

SUMMARY DNA polymerase activity was found in the cytoplasmic fraction and in isolated nuclei from anterior pituitary glands of male rats. The enzyme activity was assayed by measuring the incorporation of [3H]dTTP into DNA in a medium containing Tris-HCl buffer (pH 8·5), the four deoxyribonucleoside triphosphates, Mg2 +, ATP and activated calf thymus DNA. The DNA polymerase activity decreased with age in glands from animals aged 25 days to over a year but increased after oestrone treatment in vivo. These changes in activity, more pronounced in the cytoplasmic fraction than in the isolated nuclei, were similar to changes in DNA synthesis measured in anterior pituitary glands under the same physiological conditions. Isolated nuclei also retained endogenous DNA synthetic activity in the absence of added template. Addition of a cytoplasmic fraction to the reaction medium stimulated activity by as much as 1·9-fold but the degree of stimulation was the same whether the cytoplasm was from young, old or oestrone-treated animals.

scholarly journals The occurrence of two types of deoxyribonucleic acid polymerase activity in the main classes of nuclei obtained from the brains of infant and adult rats

1974 ◽  
Vol 140 (1) ◽  
pp. 65-71 ◽  
Author(s):  
M. A. Stambolova ◽  
D. Cox ◽  
A. P. Mathias
Keyword(s):  
Rat Brain ◽  
Dna Polymerase ◽  
Gradient Centrifugation ◽  
Polymerase Activity ◽  
Synthetic Activity ◽  
Relative Distribution ◽  
Adult Rats ◽  
Different Types ◽  
Dna Template

1. The influence of exogenous or activated DNA template on the DNA polymerase activity in the different types of intact nuclei from rat brain tissue was determined. The different amounts or physical state of the DNA template did not produce significant differences in the relative distribution of the DNA polymerase activity between the separate groups of nuclei. 2. The DNA polymerase activities, fractionated by sucrose gradient centrifugation into enzyme A and enzyme B, were found to be present in the extracts of all types of rat brain nuclei. The distribution of these two activities in the ‘particulate’ and ‘soluble’ fractions of the separate groups of nuclei from 10-day-old and adult rats was studied. The findings are related to the DNA-synthetic activity in vivo of the intact nuclei and the possible biological functions of the DNA polymerase activities are discussed.

scholarly journals DNA Polymerase and dRP-lyase activities of polymorphic variants of human Pol ι

2021 ◽  
Vol 478 (7) ◽  
pp. 1399-1412
Author(s):  
Evgeniy S. Shilkin ◽  
Anastasia S. Gromova ◽  
Margarita P. Smal ◽  
Alena V. Makarova
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
Altered Expression ◽  
Dna Polymerase Iota ◽  
Nucleotide Incorporation ◽  
Lyase Activity ◽  
Polymorphic Variants ◽  
Y Family

Y-family DNA polymerase iota (Pol ι) is involved in DNA damage response and tolerance. Mutations and altered expression level of POLI gene are linked to a higher incidence of cancer. We biochemically characterized five active site polymorphic variants of human Pol ι: R71G (rs3218778), P118L (rs554252419), I236M (rs3218784), E251K (rs3218783) and P365R (rs200852409). We analyzed fidelity of nucleotide incorporation on undamaged DNA, efficiency and accuracy of DNA damage bypass, as well as 5′-deoxyribophosphate lyase (dRP-lyase) activity. The I236M and P118L variants were indistinguishable from the wild-type Pol ι in activity. The E251K and P365R substitutions altered the spectrum of nucleotide incorporation opposite several undamaged DNA bases. The P365R variant also reduced the dRP-lyase activity and possessed the decreased TLS activity opposite 8-oxo-G. The R71G mutation dramatically affected the catalytic activities of Pol ι. The reduced DNA polymerase activity of the R71G variant correlated with an enhanced fidelity of nucleotide incorporation on undamaged DNA, altered lesion-bypass activity and reduced dRP-lyase activity. Therefore, this amino acid substitution likely alters Pol ι functions in vivo.

scholarly journals Heterotrimeric PCNA Increases the Activity and Fidelity of Dbh, a Y-family Translesion DNA Polymerase Prone to Creating Single-Base Deletion Mutations

2020 ◽  
Author(s):  
Yifeng Wu ◽  
William Jaremko ◽  
Ryan C. Wilson ◽  
Janice D. Pata
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
List Type ◽  
Single Base ◽  
Deletion Mutations ◽  
Single Base Deletion ◽  
Translesion Dna ◽  
Y Family

AbstractDbh is a Y-family translesion DNA polymerase from Sulfolobus acidocaldarius, an archaeal species that grows in harsh environmental conditions. Biochemically, Dbh displays a distinctive mutational profile, creating single-base deletion mutations at extraordinarily high frequencies (up to 50%) in specific repeat sequences. In cells, however, Dbh does not appear to contribute significantly to spontaneous frameshifts in these same sequence contexts. This suggests that either the error-prone DNA synthesis activity of Dbh is reduced in vivo and/or Dbh is restricted from replicating these sequences. Here, we test the hypothesis that the propensity for Dbh to make single base deletion mutations is reduced through interaction with the S. acidocaldarius heterotrimeric sliding clamp processivity factor, PCNA-123. We first confirm that Dbh physically interacts with PCNA-123, with the interaction requiring both the PCNA-1 subunit and the C-terminal 10 amino acids of Dbh, which contain a predicted PCNA-interaction peptide (PIP) motif. This interaction stimulates the polymerase activity of Dbh, even on short, linear primer-template DNA by increasing the rate of nucleotide incorporation. This stimulation requires an intact PCNA-123 heterotrimer and a DNA duplex length of at least 18 basepairs, the minimal length predicted from structural data to bind to both the polymerase and the clamp. Finally, we find that PCNA-123 increases the fidelity of Dbh on a single-base deletion hotspot sequence 3-fold by promoting an increase in the rate of correct, but not incorrect, nucleotide addition and propose that PCNA-123 induces Dbh to adopt a more active conformation that is less prone to creating deletions during DNA synthesis.HighlightsPCNA increases the fidelity of Dbh polymerase on a deletion-hotspot sequence.The interaction stimulates incorporation of the correct, but not incorrect, nucleotide.A minimal duplex length of 18 bp is required for PCNA to stimulate polymerase activity.Structural modeling suggests that PCNA induces a conformational change in Dbh.

scholarly journals Efficient and Error-Free Replication past a Minor-Groove N2-Guanine Adduct by the Sequential Action of Yeast Rev1 and DNA Polymerase ζ

2004 ◽  
Vol 24 (16) ◽  
pp. 6900-6906 ◽  
Author(s):  
M. Todd Washington ◽  
Irina G. Minko ◽  
Robert E. Johnson ◽  
Lajos Haracska ◽  
Thomas M. Harris ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Close Contact ◽  
Dna Polymerases ◽  
Minor Groove ◽  
Synthetic Activity ◽  
Oxidation Reactions ◽  
Lesion Bypass ◽  
Dna Minor Groove

ABSTRACT Rev1, a member of the Y family of DNA polymerases, functions in lesion bypass together with DNA polymerase ζ (Polζ). Rev1 is a highly specialized enzyme in that it incorporates only a C opposite template G. While Rev1 plays an indispensable structural role in Polζ-dependent lesion bypass, the role of its DNA synthetic activity in lesion bypass has remained unclear. Since interactions of DNA polymerases with the DNA minor groove contribute to the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, here we examine the possibility that unlike other DNA polymerases, Rev1 does not come into close contact with the minor groove of the incipient base pair, and that enables it to incorporate a C opposite the N 2-adducted guanines in DNA. To test this idea, we examined whether Rev1 could incorporate a C opposite the γ-hydroxy-1,N 2-propano-2′deoxyguanosine DNA minor-groove adduct, which is formed from the reaction of acrolein with the N 2 of guanine. Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from other oxidation reactions. We show here that Rev1 efficiently incorporates a C opposite this adduct from which Polζ subsequently extends, thereby completing the lesion bypass reaction. Based upon these observations, we suggest that an important role of the Rev1 DNA synthetic activity in lesion bypass is to incorporate a C opposite the various N 2-guanine DNA minor-groove adducts that form in DNA.

BIOSYNTHESIS OF GONADOTROPHINS BY RAT PITUITARIES IN VITRO

1975 ◽  
Vol 66 (3) ◽  
pp. 369-374 ◽  
Author(s):  
MRIDULA CHOWDHURY ◽  
EMIL STEINBERGER
Keyword(s):  
Comparative Study ◽  
Anterior Pituitary ◽  
Male Rats ◽  
Leucine Incorporation ◽  
Experimental Conditions ◽  
Rat Pituitary ◽  
Pituitary Glands ◽  
Endogenous Proteases ◽  
Pituitary Homogenate

SUMMARY A method has been developed for studying biosynthesis of FSH in the rat pituitary in vitro. Anterior pituitary glands were incubated with [3H]leucine; a specific and sensitive immunoprecipitation technique was used to isolate FSH from the pituitary homogenate. Total FSH content of the samples was measured by a double-antibody radioimmunoassay technique. Using this technique, a comparative study of LH and FSH synthesis in the same pituitary of adult male rats incubated for various intervals (0·5–6 h) was done. Increased incorporation of [3H]leucine into both LH and FSH with time was noted. The rate and amount of [3H]leucine incorporation into FSH was found to be higher than that into LH, indicating that either the rate of FSH synthesis is higher than that of LH or FSH has more leucine residues than LH. Greater susceptibility of LH to degradation by endogenous proteases during dialysis may also reflect less incorporation of [3H]leucine into LH. This method provides a reliable tool for evaluating FSH synthesis under various experimental conditions.

scholarly journals Gonadotrope Plasticity at Cellular and Population Levels

Endocrinology ◽  
2012 ◽  
Vol 153 (10) ◽  
pp. 4729-4739 ◽  
Author(s):  
Zahara Alim ◽  
Cheryl Hartshorn ◽  
Oliver Mai ◽  
Iain Stitt ◽  
Colin Clay ◽  
...  
Keyword(s):  
Pituitary Gland ◽  
Anterior Pituitary ◽  
Ex Vivo ◽  
Single Cells ◽  
Anterior Pituitary Gland ◽  
Female Mice ◽  
Model Animal ◽  
Pituitary Glands ◽  
Acute Changes

Abstract Hormone-secreting cells within the anterior pituitary gland may form organized and interdigitated networks that adapt to changing endocrine conditions in different physiological contexts. For gonadotropes, this might reflect a strategy to cope with acute changes throughout different female reproductive stages. The current study examined gonadotropes in female mice at characteristically different hormonal stages: prepubertal, postpubertal, and lactating. Gonadotrope plasticity was examined at the level of the whole population and single cells at different stages by imaging both fixed and live pituitary slices. The use of a model animal providing for the identification of selectively fluorescent gonadotropes allowed the particular advantage of defining cellular plasticity specifically for gonadotropes. In vivo analyses of gonadotropes relative to vasculature showed significantly different gonadotrope distributions across physiological states. Video microscopy studies using live slices ex vivo demonstrated pituitary cell plasticity in the form of movements and protrusions in response to GnRH. As positive feedback from rising estradiol levels is important for priming the anterior pituitary gland for the LH surge, experiments provide evidence of estradiol effects on GnRH signaling in gonadotropes. The experiments presented herein provide new insight into potential plasticity of gonadotropes within the anterior pituitary glands of female mice.

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