Workpackage 3
WP 3 Generation and Analysis Biology of Models of DNA damage response and repair mechanisms
(partners involved: 1, 3, 4, 5, 7, 8, 9, 11, 12, 13)
The models for studying genome surveillance either in mice, DT40 cell lines or yeasts compose the central part of this integrated project. Generation and analysis of mutants has been a focus of several of the collaborating groups for many years. This has resulted in the largest and most complete collection of DNA repair mutant mice in the world. Mice with disruptions in most major DNA repair pathways are available: XPA, XPC, XPB, XPD, ERCC1, CSA and CSB for NER, UNG, MAG, NTH1, OGG1 and PARP for BER, RAD54, RAD54B and RAD52 for HR, DNA-PKCS and Ku80 for NHEJ, mHR6A , mHR6B mRev1 and Rev3 for TLS,and several mutants for MMR.
In addition, several other mice are presently being generated, including mice carrying compromised cell cycle checkpoint genes. The otherwise homogeneous genetic background makes this collection uniquely fit to study (sometimes subtle) effects of the specific mutations on carcinogenesis and ageing. Especially the gene expression profiling (WP4) will depend heavily on this aspect of the mouse model collection. The work in mice will be complemented and extended with work in yeast, where the possibilities for genetic screens to uncover new repair factors are much greater. Since repair factors have been highly conserved through evolution, any new repair factor found in yeast is likely to have a mammalian counterpart which will then be the subject for knock-out cell lines and mutant mice.
Further, the collaborating labs can provide a complete collection of yeast isogenic mutants strains altered in the different replication, repair, recombination, cell cycle and checkpoint sub-pathways. Some of these backgrounds have been invaluable for the dissection of the molecular mechanisms preventing genome instability and, particularly for solving the complexity of certain specialized cellular processes coupling replication to recombination and repair to checkpoint control. The vertebrate DT40 chicken-derived cell line has a number of unique advantages. DT40 cells were obtained by immortalisation of chicken B-lymphocytes at a developmental stage at which HR activity is greatly elevated. As a result these cells have unique, “yeast-like,” advantages for genetic manipulation. Genes can be completely “knocked-out,” or site-specific mutations (suitable for detailed structure function studies) can be “knocked-in” with great facility. In addition, completely novel functions (such as epitopes suitable for rapid affinity purification or fluorescent labelling of gene products) can be applied easily by “knock-in” strategies. The elevated HR activity of these cells results in up to 2 out of 3 transformants being site specifically integrated into the DT40 genome, which compare very favourably to the very low rate of site specific integration in vertebrate systems.
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