Development of biomarkers and their application to the study of chromatin epistate effect on DNA damage and repair

Direct live cell analysis markers are very important for the better understanding of cellular processes and pathways. In this thesis we describe the development and characterization of live cell markers and their application to the study of interplay between chromatin epistate and DNA damage and repair. Initially, we did an in vivo characterization of γ-H2AX nanobody (heavy chain antibodies from alpacas) based live cell DNA double strand break marker. As so far the analysis of the post-translational modifications of histone H2AX (γ-H2AX) involved in DNA double strands breaks, was not studied in living cells, we used nanobodies as a tool. These single chain nanobodies can be genetically encoded and fused to a fluorescent tag, which can then be used to detect and trace proteins and other cellular components in vivo. After in vivo characterization, we found that epitope recognition was competed by endogenous proteins recognzing the same modification (e.g., MDC1) and suffered also from alternative binding to mimicking SQ motifs present in proteins such as XRCC1 repair factor. In parallel, a novel live cell marker for DNA replication and repair was developed by targeting the PCNA factor using cell penetrating peptide technology. PCNA is a key protein involved in DNA replication and repair pathways. Cells transfected with the fluorescent fusions of PCNA are widely used. To prevent the need for prior transfection and circumvent the problem of difficult to transfect and/or short lived cells, we developed and validated a cell permeable PCNA interacting peptide. This is the first ever attempt in developing an instantaneous biomarker, which needs only seconds to label the target. To further extend the applicability of the cell penetrating peptide system for site specific labeling of any recombinant histidine tagged as well as naturally existing histidine rich proteins in living cells, tris-nitrilotriacetic acid (NTA) probes was used. Here, we also optimized a labeling protocol so that histidine tagged proteins overexpressed in cells can be labeled with fluorescently tagged trisNTA in fixed and living cells. Finally, we explored DNA damage and repair in the context of open and closed chromatin states achieved by incorporation of different histone H2A variants (canonical H2A versus H2A.Bbd). Our established replication/repair marker was used here to test its co-localization with replication factors like PCNA. On a first set of experiments we analyzed DNA replication. Our data suggested that H2A.Bbd is enriched in the sites of active DNA replication leading to shortened S phase duration. On a second set of experiments, H2A.Bbd-GFP expressing cells were found to be more sensitive than H2A-GFP when exposed to UVC, The sensitivity was quantified by the level of cyclobutane pyrimidine dimer (CPD). Based on these results we hypothesized the reason for this increased damage could be due to more open chromatin comprising the H2A.Bbd and, on the other hand, the decreased damage due to more condensed chromatin comprising H2A. Hence, for the validation of our hypothesis we reversed the chromatin condensation states in both stable cell lines, i.e., we induced condensation of the chromatin of H2A.Bbd overexpressing cell lines and decondensation of the chromatin of H2A. overexpressing cell lines. Subsequent CPD damage analysis supported our hypothesis. The open chromatin nature leading to increased DNA damage susceptibility and shortened S-phase corelated with phenotypic characteristics of Hodgkin lymphoma cells known to overexpress H2A.Bbd.