Highly selective affinity labeling of the primer-binding site of E. coli DNA polymerase I

Membrane-associated phosphoinositidase C activity has been identified in Dictyostelium discoideum using phosphatidylinositol 4,5-bisphosphate as exogenous substrate. Maximal activity was observed with 0.4 mM phosphatidylinositol 4,5-bisphosphate at pH 7.0. The enzyme was stimulated

by micromolar concentrations of free calcium with maximal activity at 100 μM.     

The separation of subunits of phenylalanyl-tRNA-synthetase from Escherichia coli MRE-600 by means of affinity chromatography in dissociation conditions

The method of affinity chromatography on sepharose with immobilized tRNA in the presence of urea was developed for separating the subunits of phenylalanyl-tRNA synthetase from E. coli MRE-600 (subunit structure alpha 2 beta 2). Specific binding of large beta-subunits of the enzyme on immobilized tRNA testifies the localization of the tRNA-binding center on the beta-subunit of phenylalanyl-tRNA synthetase. Separately alpha- and beta-subunits of the enzyme exhibit no catalytic activity. Incubation of the mixture of alpha- and beta-subunits in conditions leading to reassociation of the oligomeric structure results in restoration of catalytic activity of the enzyme. In the presence of urea resin with immobilised analogs of ATP binds alpha- and beta-subunits of the enzyme. This testifies the presence of nucleotide-binding sites on both subunits. The possibility of using the affinity chromatography method to separate non-identical subunits of different enzymes is discussed.     

Interaction of DNA topoisomerase 1 with DNA intermediates and proteins of base excision repair

The interaction of human recombinant DNA topoisomerase 1 (Top1) with linear and circular DNA structures containing a nick or short gap but lacking a specific Top1 recognition site was studied. The effect of key excision repair proteins on formation of the Top1 covalent adduct with the DNA repair intermediates was shown. Partial inhibition of the Top1-DNA-adduct formation upon addition of poly(ADP-ribose) polymerase 1 in the absence of NAD⁺ was shown, whereas in the presence of NAD⁺ formation of a high molecular weight product, most likely corresponding to poly(ADP)-ribosylated Top1-DNA adduct, was observed. The data show that the key base excision repair proteins can influence formation of suicide Top1-DNA adducts. Top1 was identified by immunoprecipitation in the bovine testis nuclear extract as the protein forming the main modification product with nick-containing DNA.     

RPA repair recognition of DNA containing pyrimidines bearing bulky adducts

Recognition of new DNA nucleotide excision repair (NER) substrate analogs, 48-mer ddsDNA (damaged double-stranded DNA), by human replication protein A (hRPA) has been analyzed using fluorescence spectroscopy and photoaffinity modification. The aim of the present work was to find quantitative characteristics of RPA-ddsDNA interaction and RPA subunits role in this process. The designed DNA structures bear bulky substituted pyrimidine nitrogen bases at the inner positions of duplex forming DNA chains. The photoreactive 4-azido-2,5-difluoro-3- pyridin-6-yl (FAP) and fluorescent antracenyl, pyrenyl (Antr, Pyr) groups were introduced via different linker fragments into exo-4N of deoxycytidine or 5C of deoxyuridine. J-dU-containing DNA was used as a photoactive model of undamaged DNA strands. The reporter group was a fluorescein residue, introduced into the 5'-phosphate end of one duplex-forming DNA strand. RPA-dsDNA association constants and the molar RPA/dsDNA ratio have been calculated based on fluorescence anisotropy measurements under conditions of a 1:1 RPA/dsDNA molar ratio in complexes. The evident preference for RPA binding to ddsDNA over undamaged dsDNA distinctly depends on the adduct type and varies in the following way: undamaged dsDNA < Antr-dC-ddsDNA < mmdsDNA < FAPdU-, Pyr-dU-ddsDNA < FAP-dC-ddsDNA (K(D) = 68 +/- 1; 25 +/- 6; 13 +/- 1; 8 +/- 2, and 3.5 +/- 0.5 nM correspondingly) but weakly depends on the chain integrity. Interestingly the bulkier lesions not in all cases have a greater effect on RPA affinity to ddsDNA. The experiments on photoaffinity modification demonstrated only p70 of compactly arranged RPA directly interacting with dsDNA. The formation of RPA-ddsDNA covalent adducts was drastically reduced when both strands of DNA duplex contained virtually opposite located FAP-dC and Antr-dC. Thus RPA requires undamaged DNA strand presence for the effective interaction with dsDNA bearing bulky damages and demonstrates the early NER factors characteristic features underlying strand discrimination capacity and poor activity of the NER system toward double damaged DNA.     

The Effect of Erythrocyte Lysis in Selected Animals on the Absorption Spectra of Oxyhemoglobin

Biophysics - Abstract—The absorption spectra of lysed and non-lysed erythrocyte samples isolated from chicken, goose, and guinea pig blood were studied. It was found that the position of the...     

Use of Modified Flap Structures for Study of Base Excision Repair Proteins

To investigate interactions between proteins participating in the long-patch pathway of base excision repair (BER), DNA duplexes with flap strand containing modifications in sugar phosphate backbone within the flap-forming oligonucleotides were designed. When the flap-forming oligonucleotide consisted of two sequences bridged by a decanediol linker located in the flap strand near the branch point, the efficiency and position of cleavage by flap endonuclease 1 (FEN1) differed from those for natural flap. The cleavage rate of chimeric structure by FEN1 was lower than that of a normal substrate. When we introduced the second modification in the flap-forming oligonucleotide, the cleavage rate decreased significantly. To estimate efficiency of recognition and processing of the chimeric structures by BER proteins, we studied the rate of DNA synthesis by DNA polymerase beta (Pol beta) and the rate of nucleotide excision at the 3'-end of the initiating primer by apurinic/apyrimidinic endonuclease 1 (APE1) compared with those for the natural DNA duplexes. Efficiency of strand-displacement DNA synthesis catalyzed by Pol beta was shown to be higher for flap structures containing non-nucleotide linkers. The chimeric structures were processed by the 3'-exonuclease activity of APE1 with efficiency lower than that for a normal flap structure. Thus, DNA duplexes with modifications in sugar phosphate backbone can be used to mimic intermediates of the long-patch pathway of BER in reconstituted systems containing FEN1. Based on chimeric and natural oligonucleotides, photoreactive DNA structures were designed. The photoreactive dCMP moiety was introduced into the 3'-end of DNA primer via the activity of Pol beta. The photoreactive DNA duplexes--3'-recessed DNA, nicked DNA, and flap structures containing natural and chimeric oligonucleotides--were used for photoaffinity labeling of BER proteins.     

Trapping of human DNA topoisomerase I by DNA structures mimicking intermediates of DNA repair

In this report we show that human DNA Topoisomerase I (Top1) forms DNA-protein adducts with nicked and gapped DNA structures lacking a conventional Top1 cleavage site. The radioactively labeled crosslinking products were identified by SDS-gel electrophoresis. The chemical structure of the groups at 5' or 3' end of the nick does not have an effect on the formation of these covalent adducts. Therefore, all kinds of nicks, either directly induced by ionizing radiation or reactive oxygen species or indirectly induced in the course of base excision repair (BER) are targets for Top1 that competes with BER proteins and other nick-sensors. Top1-DNA covalent adducts formed in cells exposed to DNA damaging agents can promote genetic instability.     

Alterations in the nucleocytoplasmic transport in apoptosis: Caspases lead the way

Apoptosis is a mode of regulated cell death that is indispensable for the morphogenesis, development and homeostasis of multicellular organisms. Caspases are cysteine‐dependent aspartate‐specific proteases, which function as initiators and executors of apoptosis. Caspases are cytosolic proteins that can cleave substrates located in different intracellular compartments during apoptosis. Many years ago, the involvement of caspases in the regulation of nuclear changes, a hallmark of apoptosis, was documented. Accumulated data suggest that apoptosis‐associated alterations in nucleocytoplasmic transport are also linked to caspase activity. Here, we aim to discuss the current state of knowledge regarding this process. Particular attention will be focused on caspase nuclear entry and their functions in the demolition of the nucleus upon apoptotic stimuli.