Effect of proliferating cell nuclear antigen ubiquitination and chromatin structure on the dynamic properties of the Y-family DNA polymerases

Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) η and ι are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that polη is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of polη in foci, it results in an increased residence time in foci. polι is even more mobile than polη, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both polη and polι diffuse through the cell but that they are transiently immobilized for ∼150 ms, with a larger proportion of polη than polι immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of polη but not polι. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA

Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR-Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms

Xeroderma pigmentosum group A protein loads as a separate factor onto DNA lesions

Nucleotide excision repair (NER) is the main DNA repair pathway in mammals for removal of UV-induced lesions. NER involves the concerted action of more than 25 polypeptides in a coordinated fashion. The xeroderma pigmentosum group A protein (XPA) has been suggested to function as a central organizer and damage verifier in NER. How XPA reaches DNA lesions and how the protein is distributed in time and space in living cells are unknown. Here we studied XPA in vivo by using a cell line stably expressing physiological levels of functional XPA fused to green fluorescent protein and by applying quantitative fluorescence microscopy. The majority of XPA moves rapidly through the nucleoplasm with a diffusion rate different from those of other NER factors tested, arguing against a preassembled XPA-containing NER complex. DNA damage induced a transient ( approximately 5-min) immobilization of maximally 30% of XPA. Immobilization depends on XPC, indicating that XPA is not the initial lesion recognition protein in vivo. Moreover, loading of replication protein A on NER lesions was not dependent on XPA. Thus, XPA participates in NER by incorporation of free diffusing molecules in XPC-dependent NER-DNA complexes. This study supports a model for a rapid consecutive assembly of free NER factors, and a relatively slow simultaneous disassembly, after repair

Analysis of the DNA damage response in living cells

DNA lesions arising from environmental and endogenous sources induce various cellular responses including cell cycle arrest, DNA repair and apoptosis. Although detailed insights into the biochemical mechanisms and composition of DNA repair pathways have been obtained from in vitro experiments, a better understanding of the interplay and regulation of these pathways requires DNA repair studies in living cells. In this study we employed laser microirradiation and photobleaching techniques in combination with specific mutants and inhibitors to analyze the real-time accumulation of proteins at laser-induced DNA damage sites in vivo, thus unravelling the mechanisms underlying the coordination of DNA repair in living cells. The immediate and faithful recognition of DNA lesions is central to cellular survival, but how these lesions are detected within the context of chromatin is still unclear. In vitro data indicated that the DNA-damage dependent poly(ADP-ribose) polymerases, PARP-1 and PARP-2, are involved in this crucial step of DNA repair. With specific inhibitors, mutations and photobleaching analysis we could reveal a complex feedback regulated mechanism for the recruitment of the DNA damage sensor PARP-1 to microirradiated sites. Activation of PARP-1 results in localized poly(ADP-ribosyl)ation and amplifies a signal for the subsequent rapid recruitment of the loading platform XRCC1 which coordinates the assembly of the repair machinery. Using similar techniques we could demonstrate the immediate and transient binding of the RNA Polymerase II cofactor PC4 to DNA damage sites, which depended on its single strand binding capacity. This establishes an interesting link between DNA repair and transcription. We propose a role for PC4 in the early steps of the DNA damage response, recognizing and stabilizing single stranded DNA (ssDNA) and thereby facilitating DNA repair by enabling repair factors to access their substrates. After DNA lesions have been successfully detected they have to be handed over to the repair machinery which restores genome integrity. Efficient repair requires the coordinated recruitment of multiple enzyme activities which is believed to be controlled by central loading platforms. As laser microirradiation induces a variety of different DNA lesions we could directly compare the recruitment kinetics of the two loading platforms PCNA and XRCC1 which are involved in different repair pathways side by side. We could demonstrate that PCNA and XRCC1 show distinct recruitment and binding kinetics with the immediate and fast recruitment of XRCC1 preceding the slow and continuous recruitment of PCNA. Introducing consecutively multiple DNA lesions within a single cell, we further demonstrated that these different recruitment and binding characteristics have functional consequences for the capacity of PCNA and XRCC1 to respond to successive DNA damage events. To further study the role of PCNA and XRCC1 as loading platforms in DNA repair, we extended our analysis to their respective interaction partners DNA Ligase I and III. Although these DNA Ligases are highly homologous and catalyze the same enzymatic reaction, they are not interchangeable and fulfil unique functions in DNA replication and repair. With deletion and mutational analysis we could identify domains mediating the specific recruitment of DNA Ligase I and III to distinct repair pathways through their interaction with PCNA and XRCC1. We conclude that this specific targeting may have evolved to accommodate the particular requirements of different repair pathways (single nucleotide replacement vs. synthesis of short stretches of DNA) and thus enhances the efficiency of DNA repair. Interestingly, we found that other PCNA-interacting proteins exhibit recruitment kinetics similar to DNA Ligase I, indicating that PCNA not only serves as a central loading platform during DNA replication, but also coordinates the recruitment of multiple enzyme activities to DNA repair sites. Accordingly, we found that the maintenance methyltransferase DNMT1, which is known to associate with replication sites through binding to PCNA, is likewise recruited to DNA repair sites by PCNA. We propose that DNMT1, like in DNA replication, preserves methylation patterns in the newly synthesized DNA, thus contributing to the restoration of epigenetic information in DNA repair. In summary, we found immediate and transient binding of repair factors involved in DNA damage detection and signalling, while repair factors involved in the later steps of DNA repair, like damage processing, DNA ligation and restoration of epigenetic information, showed a slow and persistent accumulation at DNA damage sites. We conclude that DNA repair is not mediated by binding of a preassembled repair machinery, but rather coordinated by the sequential recruitment of specific repair factors to DNA damage sites

Inference of protein kinetics by stochastic modeling and simulation of fluorescence recovery after photobleaching experiments

Motivation: Fluorescence recovery after photobleaching (FRAP) is a functional live cell imaging technique that permits the exploration of protein dynamics in living cells. To extract kinetic parameters from FRAP data, a number of analytical models have been developed. Simplifications are inherent in these models, which may lead to inexhaustive or inaccurate exploitation of the experimental data. An appealing alternative is offered by the simulation of biological processes in realistic environments at a particle level. However, inference of kinetic parameters using simulation-based models is still limited. Results: We introduce and demonstrate a new method for the inference of kinetic parameter values from FRAP data. A small number of in silico FRAP experiments is used to construct a mapping from FRAP recovery curves to the parameters of the underlying protein kinetics. Parameter estimates from experimental data can then be computed by applying the mapping to the observed recovery curves. A bootstrap process is used to investigate identifiability of the physical parameters and determine confidence regions for their estimates. Our method circumvents the computational burden of seeking the best-fitting parameters via iterative simulation. After validation on synthetic data, the method is applied to the analysis of the nuclear proteins Cdt1, PCNA and GFPnls. Parameter estimation results from several experimental samples are in accordance with previous findings, but also allow us to discuss identifiability issues as well as cell-to-cell variability of the protein kinetics. Implementation: All methods were implemented in MATLAB R2011b. Monte Carlo simulations were run on the HPC cluster Brutus of ETH Zurich. Contact: [email protected] or [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin