[Quaternary structures of human cytoplasmic and nuclear PCNA are the same]. / Chetvertichnaia struktura iadernogo antigena deliashchikhsia kletok (PCNA) v tsitoplazme i iadre kletok cheloveka organizovana odinakovo.

Properties and mechanisms of PCNA (proliferating cell nuclear antigen) functions have been investigated for a long time and are studied in great detail. As follows from its name, most known PCNA functions (DNA replication, DNA repair, DNA recombination and others) are connected with cell proliferation and localization of this protein in nuclei. In addition, there is good reason to believe that PCNA also performs some functions in the cytoplasm. However, the possible role and mechanisms of PCNA action in the cytoplasm require careful study and clarification. Interestingly, such cells as neutrophils differ in that they are non-dividing on one hand and on the other hand contain a rather large amount of PCNA, which is localized only in the cytoplasm, that is, they are an ideal model for the study of cytoplasmic PCNA. Using cross-linkages with formaldehyde, we showed that this cytoplasmic PCNA is cross-linked in a similar way, that is, organized in the same way as the nuclear PCNA that is present in the proliferating cells. Previously, we showed that PCNA in such cells is organized into a dynamic complex of double trimer on the basis of the back-to-back principle (Naryzhny S.N. et al. (2005) J. Biol. Chem., 280, 13888). Apparently, such organization of this hub-protein allows it to better coordinate the processes taking place in the cytoplasm as well.

[Experimental estimation of proteome size for cells and human plasma].

Huge range of concentrations of different protein and insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of human proteome. In our investigations, we tried to evaluate the size of different proteomes (cells and plasma). The approach used is based on detection of protein spots in 2-DE after staining by protein dyes with different sensitivities. The function representing the dependence of the number of protein spots on sensitivity of protein dyes was generated. Next, by extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) it was calculated that a single human cell (HepG2) may contain minimum 70,000 proteoforms, and plasma--1.5 mln. Utilization of this approach to other, smaller proteomes showed the competency of this extrapolation. For instance, the size of mycoplas ma (Acholeplasma laidlawii) was estimated in 1100 proteoforms, yeast (Saccharomyces cerevisiae)--40,000, E. coli--6200, P. furiosus--3400. In hepatocytes, the amount of proteoforms was the same as in HepG2--70,000. Significance of obtained data is in possibilities to estimating the proteome organization and planning next steps in its study.

Autonomous 3'-->5' exonucleases can proofread for DNA polymerase beta from rat liver.

Autonomous 3'-->5'exonucleases are not bound covalently to DNA polymerases but are often involved in replicative complexes. Such exonucleases from rat liver, calf thymus and Escherichia coli (molecular masses of 28+/-2 kDa) are shown to increase more than 10-fold the accuracy of DNA polymerase beta (the most inaccurate mammalian polymerase) from rat liver in the course of reduplication of the primed DNA of bacteriophage phiX174 amber 3 in vitro. The extent of correction increases together with the rise in 3'-->5' exonuclease concentration. Extrapolation of the in vitro DNA replication fidelity to the cellular levels of rat exonuclease and beta-polymerase suggests that exonucleolytic proofreading could augment the accuracy of DNA synthesis by two orders of magnitude. These results are not explained by exonucleolytic degradation of the primers ("no synthesis-no errors"), since similar data are obtained with the use of the primers 15 or 150 nucleotides long in the course of a fidelity assay of DNA polymerases, both alpha and beta, in the presence of various concentrations of 3'-->5' exonuclease.

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.