http://repub.eur.nl/res/aut/11104/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/3202/ 2002-10-01T00:00:00Z Homologous recombination is one of the major pathways for repair of DNA double-strand breaks (DSBs). Important proteins in this pathway are Rad51 and Rad54. Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) that mediates pairing with and strand invasion of homologous duplex DNA with the assist of Rad54. We estimated that the nucleus of a mouse embryonic stem (ES) cells contains on average 4.7x10(5) Rad51 and 2.4x10(5) Rad54 molecules. Furthermore, we showed that the amount of Rad54 was subject to cell cycle regulation. We discuss our results with respect to two models that describe how Rad54 stimulates Rad51-mediated DNA strand invasion. The models differ in whether Rad54 functions locally or globally. In the first model, Rad54 acts in cis relative to the site of strand invasion. Rad54 coats the Rad51 nucleoprotein filament in stoichiometric amounts and binds to the target duplex DNA at the site that is homologous to the ssDNA in the Rad51 nucleoprotein filament. Subsequently, it promotes duplex DNA unwinding. In the second model, Rad54 acts in trans relative to the site of strand invasion. Rad54 binds duplex DNA distant from the site that will be unwound. Translocation of Rad54 along the duplex DNA increases superhelical stress thereby promoting duplex DNA unwinding. http://repub.eur.nl/res/pub/31852/ 2002-01-16T00:00:00Z The aim of this thesis is to investigate mammalian DNA interstrand cross-link (ICL) repair. ICLs are formed by a number of agents used in tumor therapy, like mitomycin C and cisplatin. They constitute one of the most toxic damages to DNA, as they inhibit DNA strand separation. However, little is known about the mechanisms of !CL repair. A number of DNA repair pathways exist, each involved in the repair of specific types of DNA damage that continuously threaten cellular function. An intriguing aspect of ICL repair is the involvement of several of these repair pathways, mainly nucleotide excision repair, homologous recombination, and postreplication/translesion repair. An overview of the involvement of these different repair pathways in ICL repair is given in Chapter 4. This chapter also depicts putative models describing the co-operation of these pathways in repairing ICLs. Next to genes involved in several repair pathways, other genes have been isolated that are exclusively involved in ICL repair, such as yeast SNMJ. snml mutant yeast cells are sensitive to a number of ICL agents, but they are hardly or not sensitive to other DNAdamaging agents. As described in Chapter 5, we have investigated the human and mouse homologs of Snml. We isolated mouse SNMJ and made embryonic stem cells and mice deficient for SNMJ. Both cells and mice are viable and sensitive to mitomycin C. These results indicate that mammalian Snm 1 is involved in the cellular response to at least some types of!CLs. We also showed that Snml is probably not involved in the homologous recombination pathway of ICL repair, as two parameters for homologous recombination, the formation of mitomycin C-induced Rad51 foci and sister chromatid exchanges, are not affected in SNMJ-deficient mouse cells. http://repub.eur.nl/res/pub/9508/ 2001-01-01T00:00:00Z - DNA double-strand breaks (DSBs) in eukaryotic cells can be repaired by non-homologous end-joining or homologous recombination. The complex containing the Mre11, Rad50 and Nbs1 proteins has been implicated in both DSB repair pathways, even though they are mechanistically different. To get a better understanding of the properties of the human Mre11 (hMre11) protein, we investigated some of its biochemical activities. We found that hMre11 binds both double- and single-stranded (ss)DNA, with a preference for ssDNA. hMre11 does not require DNA ends for efficient binding. Interestingly, hMre11 mediates the annealing of complementary ssDNA molecules. In contrast to the annealing activity of the homologous recombination protein hRad52, the activity of hMre11 is abrogated by the ssDNA binding protein hRPA. We discuss the possible implications of the results for the role(s) of hMre11 in both DSB repair pathways. http://repub.eur.nl/res/pub/9605/ 2001-01-01T00:00:00Z DNA double-strand breaks (DSBs) in eukaryotic cells can be repaired by non-homologous end-joining or homologous recombination. The complex containing the Mre11, Rad50 and Nbs1 proteins has been implicated in both DSB repair pathways, even though they are mechanistically different. To get a better understanding of the properties of the human Mre11 (hMre11) protein, we investigated some of its biochemical activities. We found that hMre11 binds both double- and single-stranded (ss)DNA, with a preference for ssDNA. hMre11 does not require DNA ends for efficient binding. Interestingly, hMre11 mediates the annealing of complementary ssDNA molecules. In contrast to the annealing activity of the homologous recombination protein hRad52, the activity of hMre11 is abrogated by the ssDNA binding protein hRPA. We discuss the possible implications of the results for the role(s) of hMre11 in both DSB repair pathways. http://repub.eur.nl/res/pub/3176/ 2000-01-01T00:00:00Z DNA interstrand cross-links (ICLs) represent lethal DNA damage, because they block transcription, replication, and segregation of DNA. Because of their genotoxicity, agents inducing ICLs are often used in antitumor therapy. The repair of ICLs is complex and involves proteins belonging to nucleotide excision, recombination, and translesion DNA repair pathways in Escherichia coli, Saccharomyces cerevisiae, and mammals. We cloned and analyzed mammalian homologs of the S. cerevisiae gene SNM1 (PSO2), which is specifically involved in ICL repair. Human Snm1, a nuclear protein, was ubiquitously expressed at a very low level. We generated mouse SNM1(-/-) embryonic stem cells and showed that these cells were sensitive to mitomycin C. In contrast to S. cerevisiae snm1 mutants, they were not significantly sensitive to other ICL agents, probably due to redundancy in mammalian ICL repair and the existence of other SNM1 homologs. The sensitivity to mitomycin C was complemented by transfection of the human SNM1 cDNA and by targeting of a genomic cDNA-murine SNM1 fusion construct to the disrupted locus. We also generated mice deficient for murine SNM1. They were viable and fertile and showed no major abnormalities. However, they were sensitive to mitomycin C. The ICL sensitivity of the mammalian SNM1 mutant suggests that SNM1 function and, by implication, ICL repair are at least partially conserved between S. cerevisiae and mammals. http://repub.eur.nl/res/pub/9309/ 2000-01-01T00:00:00Z Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54 wild-type and knockout cells revealed direct evidence for a role of mRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence of mRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA. http://repub.eur.nl/res/pub/9383/ 2000-01-01T00:00:00Z DNA interstrand cross-links (ICLs) represent lethal DNA damage, because they block transcription, replication, and segregation of DNA. Because of their genotoxicity, agents inducing ICLs are often used in antitumor therapy. The repair of ICLs is complex and involves proteins belonging to nucleotide excision, recombination, and translesion DNA repair pathways in Escherichia coli, Saccharomyces cerevisiae, and mammals. We cloned and analyzed mammalian homologs of the S. cerevisiae gene SNM1 (PSO2), which is specifically involved in ICL repair. Human Snm1, a nuclear protein, was ubiquitously expressed at a very low level. We generated mouse SNM1(-/-) embryonic stem cells and showed that these cells were sensitive to mitomycin C. In contrast to S. cerevisiae snm1 mutants, they were not significantly sensitive to other ICL agents, probably due to redundancy in mammalian ICL repair and the existence of other SNM1 homologs. The sensitivity to mitomycin C was complemented by transfection of the human SNM1 cDNA and by targeting of a genomic cDNA-murine SNM1 fusion construct to the disrupted locus. We also generated mice deficient for murine SNM1. They were viable and fertile and showed no major abnormalities. However, they were sensitive to mitomycin C. The ICL sensitivity of the mammalian SNM1 mutant suggests that SNM1 function and, by implication, ICL repair are at least partially conserved between S. cerevisiae and mammals.