Nazarov, I. B., Smirnova, A. N., Krutilina, R. I., Svetlova, M. P., Solovjeva, L. V., Nikiforov, A. A., Oei, S-L., Zalenskaya, I. A., Yau, P. M., Bradbury, E. M. and Tomilin, N. V. Dephosphorylation of Histone γ-H2AX during Repair of DNA Double-Strand Breaks in Mammalian Cells and its Inhibition by Calyculin A. Radiat. Res. 160, 309–317 (2003).The induction of DNA double-strand breaks (DSBs) by ionizing radiation in mammalian chromosomes leads to the phosphorylation of Ser-139 in the replacement histone H2AX, but the molecular mechanism(s) of the elimination of phosphorylated H2AX (called γ-H2AX) from chromatin in the course of DSB repair remains unknown. We showed earlier that γ-H2AX cannot be replaced by exchange with free H2AX, suggesting the direct dephosphorylation of H2AX in chromatin by a protein phosphatase. Here we studied the dynamics of dephosphorylation of γ-H2AX in vivo and found that more than 50% was dephosphorylated in 3 h, but a significant amount of γ-H2AX could be detected even 6 h after the induction of DSBs. At this time, a significant fraction of the γ-H2AX nuclear foci co-localized with the foci of RAD50 protein that did not co-localize with replication sites. However, γ-H2AX could be detected in some cells treated with methyl methanesulfonate which accumulated RAD18 protein at stalled replication sites. We also found that calyculin A inhibited early elimination of γ-H2AX and DSB rejoining in vivo and that protein phosphatase 1 was able to remove phosphate groups from γ-H2AX-containing chromatin in vitro. Our results confirm the tight association between DSBs and γ-H2AX and the coupling of its in situ dephosphorylation to DSB repair.

Peroxiredoxins belong to a family of antioxidant proteins that neutralize reactive oxygen species. One member of this family, peroxiredoxin I (PRDX1), suppresses DNA oxidation. Peroxiredoxin V (PRDX5) has been cloned as a transcriptional corepressor, as a peroxisomal/mitochondrial antioxidant protein, and as an inhibitor of p53‐dependent apoptosis. Promoters of mammalian PRDX5 genes contain clusters of antioxidant response elements, which can bind the transcription factor NRF2. However, we found that expression of the human PRDX5 gene in situ was not stimulated by the oxidative agent menadione. Silencing of the NRF2 gene in the absence of oxidative stress by specific siRNA did not decrease PRDX5 protein concentration. We also constructed clones of human lung epithelial cells A549 with siRNA‐mediated knockdown of the PRDX5 gene. This led to a significant increase in 8‐oxoguanine formation in cell DNA. In the PRDX5 knockdown clone, an increase in transcripts containing sequences of alpha‐satellite and satellite III DNAs was also detected, suggesting that this protein may be required for silencing of heterochromatin. Together, these results suggest that constitutively expressed PRDX5 gene plays an important role in protecting the genome against oxidation and may also be involved in the control of transcription of noncoding DNA.

We aimed to explore whether variants of SARS-CoV-2 (Chinese-derived strain (D614, lineage A), Italian strain PV10734 (D614G, lineage B.1.1) and Alpha strain (lineage B.1.1.7)) were able to infect monocytes (MN) and monocyte-derived macrophages (MDM) and whether these infected cells may, in turn, be vectors of infection. For this purpose, we designed an in vitro study following the evolution of MN and MDM infection at different time points in order to confirm whether these cells were permissive for SARS-CoV-2 replication. Finally, we investigated whether, regardless of viral replication, the persistent virus can be transferred to non-infected cells permissive for viral replication. Thus, we co-cultured the infected MN/MDM with permissive VERO E6 cells verifying the viral transmission. This is a further in vitro demonstration of the important role of MN and MDM in the dissemination of SARS-CoV-2 and evolution of the COVID-19 disease.

Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli’s zebra and Grevy’s zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.