Poly polymerases

Rosemary Phytopolyphenols Cause Cleavage of Poly-Polymerase and DFF45/ICAD... 

Poly(L-malate) decomposes spontaneously to L-ma-late by ester hydrolysis [2,4,5]. Hydrolytic degradation of the polymer sodium salt at pH 7.0 and 37°C results in a random cleavage of the polymer, the molecular mass decreasing by 50% after a period of 10 h [2]. The rate of hydrolysis is accelerated in acidic and alkaline solutions. This was first noted by changes in the activity of the polymer to inhibit DNA polymerase a of P. polycephalum [4]. The explanation of this phenomenon was that the degradation was slowest between pH 5-9 (Fig. 2) as would be expected if it were acid/base-catalyzed. In choosing a buffer, one should be aware of specific buffer catalysis. We found that the polymer was more stable in phosphate buffer than in Tris/HCl-buffer. 

Figure 2 Stability of /3-poly(L-malate) measured by its activity to inhibit purified DNA polymerase a of P. polyceph-alum. The relative degree of inhibition is shown (100 rel. units refer to complete inhibition). The DNA polymerase assay was carried out in the presence of 5 /tg/ml /S-poly(L-malate) as described [4]. The polymer was preincubated for 7 days at 4°C in the following buffer solutions (50 mM) KCl/HCl (—A—). Citrate (—V—). 2-(A/-Morpholino)-ethanesulfonic acid, sodium salt (—O—). Sodium phosphate (— —). N-(2-Hydroxyethyl)piperazine-N -(2-ethanesul-fonic acid), sodium salt (— — ). N,N-b s (2-Hydroxyethyl)-glycine, sodium salt (—T—). Tris/HCl (— —). 3-(Cyclo-hexylamino)-l-propanesulfonic acid, sodium salt (— —).

Poly(A) tails are added to the S end of mRNA molecules in a posttranscriptional processing step. The mRNA is first cleaved about 20 nucleotides downstream from an AAUAA recognition sequence. Another enzyme, poly(A) polymerase, adds a poly(A) tail which is subsequently extended to as many as 200 A residues. The poly(A) tail appears to protect the S end of mRNA from S —> S exonuclease attack. The presence or absence of the poly(A) tail does not determine whether a precursor molecule in the nucleus appears in the cytoplasm, because all poly(A)-tailed hnRNA molecules do not contribute to cytoplasmic mRNA, nor do all cytoplasmic mRNA molecules contain poly(A) tails... 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

Under certain circumstances DNA has both primer and template activities. For example, the addition of mononucleotides is to the 3 end of the growing DNA primer. This presents a problem with regard to how the other strand is synthesized. Biochemists have looked hard but unsuccessfully for an enzyme that can add deoxyribonucleotides onto the 5 end of DNA primers. Such a primer should contain a triphosphate on the hydroxyl group of the 5 end. Although a very active 5 -exonuclease, actually part of DNA polymerase I, has made the search for such an activated 5 end extremely difficult, investigators conclude that a polymerase able to use such a primer probably does not exist. On the contrary, good evidence suggests that the synthesis of both strands is by the known DNA poly-merases. 

In the synthesis route from acetyl-CoA to poly(3HB), at least three steps and three enzymes are involved (Fig. 1). The first step is catalyzed by the 3-keto-thiolase (EC 2.3.1.9) which reversibly links two acetyl-CoA moieties to aceto-acetyl-CoA in a Claisen-condensation. The conversion of acetoacetyl-CoA into D-(-)-3-hydroxybutyryl-CoA can be mediated by a reductase (step 2) or via a sequence catalyzed by a reductase (step 4) and two hydratases (steps 5,6). The last step, i.e., the polymerization, is catalyzed by a polymerase (step 3). This... 

Ame J-C et al. PARP-2, A novel mammalian DNA damage-dependent poly(ADP-ribose) polymerase. J Biol Chem 1999 274 17860-17868. 

Jun G et al. Ethnic differences in poly CADP-ribose) polymerase pseudogene genotype distribution association with lung cancer risk. Carcinogenesis 1999 20 1465-1469. 

Variations The poly(A) tailing kit (Ambion) produces a mRNA population with varying lengths of poly(A) tails, controlled by altering poly(A) polymerase concentrations and incubation times. An alternate method to incorporate a poly(A) tail is to clone a defined stretch of adenosines/ thymidines into the > UTR of the template pDNA. To allow transcripts to finish on an adenosine, the insert should be followed by a restriction site for an enzyme that cleaves 5 of the last antisense strand thymidine, such as Nsi I. In this way, the poly (A) tail can be incorporated directly into the... 

Template availability Methylation Exposure of DNA Attraction for RNA polymerase Promoter TATA, CAT Transcription factors Alternative Poly(A) tailing Alternative splicing Alternative translation start... 

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