markdownabstract__Abstract__ The crucial factor for the survival of an organism resides in geneti... more markdownabstract__Abstract__ The crucial factor for the survival of an organism resides in genetic stability. In fact the integrity of the DNA sequence, that carries out and regulates genetic information, can be impaired by inaccurate maintenance processes, endogenous metabolites or exogenous agents. Therefore, a sophisticated network of efficient DNA damage response mechanisms, including DNA repair, checkpoint mechanisms and damage tolerance processes, are dedicated to remove or bypass most of these genomic insults and is known as the DNA Damage Response (DDR). The relevance of this multi-protein DDR response is illustrated by the severe clinical symptoms associated with inherited defects in the DDR factors. Chapter 1 introduces the different DNA lesions, the DDR response and the most appropriate technical approach to study the different DDR factors. The various DDR associated diseases are also presented in this chapter.
The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its cru... more The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that RPA, in contrast to other replication factors, does not cluster into replication foci, which is explained by its short residence time at ssDNA. In addition to replication, RPA also plays a crucial role in both the pre- and post-incision steps of nucleotide excision repair (NER). Pre-incision factors like XPC and TFIIH accumulate rapidly at locally induced UV-damage and remain visible up to 4h. However, RPA did not reach its maximum accumulation level until 3h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8h, probably reflecting its role during the post-incision step of NER. During the pre-incision steps of NER, RPA could only be visualized at DNA lesions in incision deficient XP-F cells, however without a substantial ...
To understand how multiprotein complexes assemble and function on chromatin, we combined quantita... more To understand how multiprotein complexes assemble and function on chromatin, we combined quantitative analysis of the mammalian nucleotide excision DNA repair (NER) machinery in living cells with computational modeling. We found that individual NER components exchange within tens of seconds between the bound state in repair complexes and the diffusive state in the nucleoplasm, whereas their net accumulation at repair sites evolves over several hours. Based on these in vivo data, we developed a predictive kinetic model for the assembly and function of repair complexes. DNA repair is orchestrated by the interplay of reversible protein-binding events and progressive enzymatic modifications of the chromatin substrate. We demonstrate that faithful recognition of DNA lesions is time consuming, whereas subsequently, repair complexes form rapidly through random and reversible assembly of NER proteins. Our kinetic analysis of the NER system reveals a fundamental conflict between specificity ...
Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the ... more Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5 0 to the lesion by ERCC1-XPF and 3 0 to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5 0 incision by ERCC1-XPF precedes the 3 0 incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a 'cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity.
Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) ... more 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 ope...
Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre... more Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3′ termini. The kinetic studies are consistent with...
Influence of the live cell DNA marker DRAQ5 on chromatin-associated processes. DNA Repair, 9 (7).... more Influence of the live cell DNA marker DRAQ5 on chromatin-associated processes. DNA Repair, 9 (7). pp. 848-855.
Chromatin modifications are an important component of the of DNA damage response (DDR) network th... more Chromatin modifications are an important component of the of DNA damage response (DDR) network that safeguard genomic integrity. Recently, we demonstrated nucleotide excision repair (NER)–dependent histone H2A ubiquitination at sites of ultraviolet (UV)-induced DNA damage. In this study, we show a sustained H2A ubiquitination at damaged DNA, which requires dynamic ubiquitination by Ubc13 and RNF8. Depletion of these enzymes causes UV hypersensitivity without affecting NER, which is indicative of a function for Ubc13 and RNF8 in the downstream UV–DDR. RNF8 is targeted to damaged DNA through an interaction with the double-strand break (DSB)–DDR scaffold protein MDC1, establishing a novel function for MDC1. RNF8 is recruited to sites of UV damage in a cell cycle–independent fashion that requires NER-generated, single-stranded repair intermediates and ataxia telangiectasia–mutated and Rad3-related protein. Our results reveal a conserved pathway of DNA damage–induced H2A ubiquitination f...
Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the ... more Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5 0 to the lesion by ERCC1-XPF and 3 0 to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5 0 incision by ERCC1-XPF precedes the 3 0 incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a 'cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity.
Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) ... more Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) and 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 pol 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 pol in foci, it results in an increased residence time in foci. pol is even more mobile than pol, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both pol and pol diffuse through the cell but that they are transiently immobilized for ϳ150 ms, with a larger proportion of pol than pol immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of pol but not pol. 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. ‡ Present address:
In the last decade, live cell fluorescence microscopy experiments have revolutionized cellular an... more In the last decade, live cell fluorescence microscopy experiments have revolutionized cellular and molecular biology, enabling the localization of proteins within cellular compartments to be analysed and to determine kinetic parameters of enzymatic reactions in living nuclei to be measured. Recently, in vivo DNA labelling by DNA-stains such as DRAQ5, has provided the opportunity to measure kinetic reactions of GFP-fused proteins in targeted areas of the nucleus with different chromatin compaction levels. To verify the suitability of combining DRAQ5-staining with protein dynamic measurements, we have tested the cellular consequences of DRAQ5 DNA intercalation. We show that DRAQ5 intercalation rapidly modifies both the localization and the mobility properties of several DNA-binding proteins such as histones, DNA repair, replication and transcription factors, by stimulating a release of these proteins from their substrate. Most importantly, the effect of DRAQ5 on the mobility of essential cellular enzymes results in a potent inhibition of the corresponding cellular functions. From these observations, we suggest that great caution must be used when interpreting live cell data obtained using DRAQ5.
Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre... more Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre-and post-incision complexes. The post-incision step of NER includes gap fi lling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of factors involved are not well understood. Using immunofl uorescence, chromatin immunoprecipitation and live cell protein dynamic studies we show that RFC is implicated in post-incision NER in mammalian cells. siRNA mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment, yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3' termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5' phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA.
markdownabstract__Abstract__ The crucial factor for the survival of an organism resides in geneti... more markdownabstract__Abstract__ The crucial factor for the survival of an organism resides in genetic stability. In fact the integrity of the DNA sequence, that carries out and regulates genetic information, can be impaired by inaccurate maintenance processes, endogenous metabolites or exogenous agents. Therefore, a sophisticated network of efficient DNA damage response mechanisms, including DNA repair, checkpoint mechanisms and damage tolerance processes, are dedicated to remove or bypass most of these genomic insults and is known as the DNA Damage Response (DDR). The relevance of this multi-protein DDR response is illustrated by the severe clinical symptoms associated with inherited defects in the DDR factors. Chapter 1 introduces the different DNA lesions, the DDR response and the most appropriate technical approach to study the different DDR factors. The various DDR associated diseases are also presented in this chapter.
The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its cru... more The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that RPA, in contrast to other replication factors, does not cluster into replication foci, which is explained by its short residence time at ssDNA. In addition to replication, RPA also plays a crucial role in both the pre- and post-incision steps of nucleotide excision repair (NER). Pre-incision factors like XPC and TFIIH accumulate rapidly at locally induced UV-damage and remain visible up to 4h. However, RPA did not reach its maximum accumulation level until 3h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8h, probably reflecting its role during the post-incision step of NER. During the pre-incision steps of NER, RPA could only be visualized at DNA lesions in incision deficient XP-F cells, however without a substantial ...
To understand how multiprotein complexes assemble and function on chromatin, we combined quantita... more To understand how multiprotein complexes assemble and function on chromatin, we combined quantitative analysis of the mammalian nucleotide excision DNA repair (NER) machinery in living cells with computational modeling. We found that individual NER components exchange within tens of seconds between the bound state in repair complexes and the diffusive state in the nucleoplasm, whereas their net accumulation at repair sites evolves over several hours. Based on these in vivo data, we developed a predictive kinetic model for the assembly and function of repair complexes. DNA repair is orchestrated by the interplay of reversible protein-binding events and progressive enzymatic modifications of the chromatin substrate. We demonstrate that faithful recognition of DNA lesions is time consuming, whereas subsequently, repair complexes form rapidly through random and reversible assembly of NER proteins. Our kinetic analysis of the NER system reveals a fundamental conflict between specificity ...
Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the ... more Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5 0 to the lesion by ERCC1-XPF and 3 0 to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5 0 incision by ERCC1-XPF precedes the 3 0 incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a 'cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity.
Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) ... more 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 ope...
Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre... more Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3′ termini. The kinetic studies are consistent with...
Influence of the live cell DNA marker DRAQ5 on chromatin-associated processes. DNA Repair, 9 (7).... more Influence of the live cell DNA marker DRAQ5 on chromatin-associated processes. DNA Repair, 9 (7). pp. 848-855.
Chromatin modifications are an important component of the of DNA damage response (DDR) network th... more Chromatin modifications are an important component of the of DNA damage response (DDR) network that safeguard genomic integrity. Recently, we demonstrated nucleotide excision repair (NER)–dependent histone H2A ubiquitination at sites of ultraviolet (UV)-induced DNA damage. In this study, we show a sustained H2A ubiquitination at damaged DNA, which requires dynamic ubiquitination by Ubc13 and RNF8. Depletion of these enzymes causes UV hypersensitivity without affecting NER, which is indicative of a function for Ubc13 and RNF8 in the downstream UV–DDR. RNF8 is targeted to damaged DNA through an interaction with the double-strand break (DSB)–DDR scaffold protein MDC1, establishing a novel function for MDC1. RNF8 is recruited to sites of UV damage in a cell cycle–independent fashion that requires NER-generated, single-stranded repair intermediates and ataxia telangiectasia–mutated and Rad3-related protein. Our results reveal a conserved pathway of DNA damage–induced H2A ubiquitination f...
Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the ... more Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5 0 to the lesion by ERCC1-XPF and 3 0 to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5 0 incision by ERCC1-XPF precedes the 3 0 incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a 'cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity.
Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) ... more Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) and 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 pol 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 pol in foci, it results in an increased residence time in foci. pol is even more mobile than pol, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both pol and pol diffuse through the cell but that they are transiently immobilized for ϳ150 ms, with a larger proportion of pol than pol immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of pol but not pol. 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. ‡ Present address:
In the last decade, live cell fluorescence microscopy experiments have revolutionized cellular an... more In the last decade, live cell fluorescence microscopy experiments have revolutionized cellular and molecular biology, enabling the localization of proteins within cellular compartments to be analysed and to determine kinetic parameters of enzymatic reactions in living nuclei to be measured. Recently, in vivo DNA labelling by DNA-stains such as DRAQ5, has provided the opportunity to measure kinetic reactions of GFP-fused proteins in targeted areas of the nucleus with different chromatin compaction levels. To verify the suitability of combining DRAQ5-staining with protein dynamic measurements, we have tested the cellular consequences of DRAQ5 DNA intercalation. We show that DRAQ5 intercalation rapidly modifies both the localization and the mobility properties of several DNA-binding proteins such as histones, DNA repair, replication and transcription factors, by stimulating a release of these proteins from their substrate. Most importantly, the effect of DRAQ5 on the mobility of essential cellular enzymes results in a potent inhibition of the corresponding cellular functions. From these observations, we suggest that great caution must be used when interpreting live cell data obtained using DRAQ5.
Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre... more Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre-and post-incision complexes. The post-incision step of NER includes gap fi lling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of factors involved are not well understood. Using immunofl uorescence, chromatin immunoprecipitation and live cell protein dynamic studies we show that RFC is implicated in post-incision NER in mammalian cells. siRNA mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment, yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3' termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5' phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA.
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Papers by Audrey Gourdin