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Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that ...

University of São Paulo

During DNA replication, forks may encounter unrepaired lesions that hamper DNA synthesis. Cells have universal strategies to promote damage bypass allowing cells to survive. DNA damage tolerance can be performed upon template switch or by specialized DNA polymerases, known as translesion (TLS) polymerases. Human cells count on more than eleven TLS polymerases and this work reviews the functions of some of these enzymes: Rev1, Pol η, Pol ι, Pol κ, Pol θ and Pol ζ. The mechanisms of damage bypass vary according to the lesion, as well as to the TLS polymerases available, and may occur directly at the fork during replication. Alternatively, the lesion may be skipped, leaving a single-stranded DNA gap that will be replicated later. Details of the participation of these enzymes are revised for the replication of damaged template. TLS polymerases also have functions in other cellular processes. These include involvement in somatic hypermutation in immunoglobulin genes, direct participation in recombination and repair processes, and contributing to replicating noncanonical DNA structures. The importance of DNA damage replication to cell survival is supported by recent discoveries that certain genes encoding TLS polymerases are induced in response to DNA damaging agents, protecting cells from a subsequent challenge to DNA replication. We retrace the findings on these genotoxic (adaptive) responses of human cells and show the common aspects with the SOS responses in bacteria. Paradoxically, although TLS of DNA damage is normally an error prone mechanism, in general it protects from carcinogenesis, as evidenced by increased tumorigenesis in xeroderma pigmentosum variant patients, who are deficient in Pol η. As these TLS polymerases also promote cell survival, they constitute an important mechanism by which cancer cells acquire resistance to genotoxic chemotherapy. Therefore, the TLS polymerases are new potential targets for improving therapy against tumors.

Filling gaps in translesion DNA synthesis in human cells.

Trypanosoma cruzi is the etiologic agent of Chagas' disease. Infected cells with T. cruzi activate several responses that promote unbalance of reactive oxygen species (ROS) that may cause DNA damage that activate cellular responses including DNA repair processes. In this work, HeLa cells and AC16 human cardiomyocyte cell line were infected with T. cruzi to investigate host cell responses at genome level during parasites intracellular life cycle. In fact, alkaline sensitive sites and oxidized DNA bases were detected in the host cell genetic material particularly in early stages of infection. These DNA lesions were accompanied by phosphorylation of the histone H2Ax, inducing γH2Ax, a marker of genotoxic stress. Moreover, Poly [ADP-ribose] polymerase-1 (PARP1) and 8-oxoguanine glycosylase (OGG1) are recruited to host cell nuclei, indicating activation of the DNA repair process. In infected cells, chromatin-associated proteins are carbonylated, as a possible consequence of oxidative stress and the nuclear factor erythroid 2-related factor 2 (NRF2) is induced early after infection, suggesting that the host cell antioxidant defenses are activated. However, at late stages of infection, NRF2 is downregulated. Interestingly, host cells treated with glutathione precursor, N-acetyl cysteine, NRF2 activator (Sulforaphane), and also Benznidonazol (BNZ) reduce parasite burst significantly, and DNA damage. These data indicate that the balance of oxidative stress and DNA damage induction in host cells may play a role during the process of infection itself, and interference in these processes may hamper T. cruzi infection, revealing potential target pathways for the therapy support.

Davi J. Martins

Department of Microbiology,
Institute of Biomedical Sciences,
Department of Microbiology, Institute of Biomedical Sciences

University of São Paulo

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

Davi J. Martins

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microbiology,Institute of Biomedical Sciences,Department of Microbiology, Institute of Biomedical Sciences

University of São Paulo

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

Department of Microbiology,
Institute of Biomedical Sciences,
Department of Microbiology, Institute of Biomedical Sciences