Análisis de la deficiencia de la DNA polimerasa [mu] en la unión de extremos no homólogos. Implicaciones en envejecimiento y desarrollo tumoral

Resumen

Double strand breaks (DSBs) are the most deleterious form of DNA damage. They can lead to genomic instability and cell death (or senescence), which might eventually cause cancer and aging. Organisms have developed two main strategies to fight against DNA damage: DNA repair mechanisims and cell cycle control checkpoints. Both of them are induced by DNA damage sensors, which activate not only the initiating repair factors but also the central protein in the checkpoints control: the tumor supressor p53. The levels of activate P53, depend on amount of DNA damage, and determine the outcome of the cellular decision to undergo apoptosis, senescence or continue through the cell cycle. Consistently with the role of p53 in tumor suppression, p53-/- mice are strongly cancer prone and succumb to aggressive thymic lymphomas at an early age. The two principal mechanisms for DSBs repair are Homologous Recombination (HR) and Non Homologous End Joining (NHEJ). HR is characterized by its fidelity because it makes use of the homologous sequence to repair the damaged DNA. Therefore, HR acts mainly in the G2M phase of cell cycle, where the sister chromatids are available to act as a template. NHEJ is able to act throughout the cell cycle, mainly in S-G1 phases, and does not use large regions of homology to repair DNA breaks. However, this mechanism is more error prone. NHEJ can repair many diferent types of DSBs, even those that need end processing prior to ligation of the break. In the latter case, a polymerase activity is normaly required. Mice lacking any of the NHEJ core factors (Ku70, Ku80, DNA-PKcs, LigIV, Xrcc4 and XLF) show immunodeficiency due to blocked recombination of gene segments in B and T lymphocytes receptors (a NHEJ mediated process). These mice also exhibit ionizing radiation sensitivity and some of them (Ku80-/- and DNA-PKcs-/-) have premature aging phenotypes. NHEJ deficient mouse embryonic fibroblasts (MEFs) enter senescence prematurely due to persistent DNA damage and chromosomal instability. The combined deficiency of a NHEJ core factor and p53 results in pro-B lymphoma development, due to the survival of chromosomically aberrant lymphocytes, many of which carry specific translocations between the IgH enhancer locus and C-myc . Polymerase mu (pol ¿) is an error-prone DNA polymerase which belongs to the PolX family. Pol ¿ has high estructural similarity to TdT and, like this protein, it has terminal transferase activity. TdT¿s activity is limited to programmed rearrangements in lymphocyte development. In vitro, pol ¿ participates in specific NHEJ reactions that require DNA polymerase activity. The only in vivo role of pol ¿ that had been demonstrated before 2009 (when part of the present work was published: Lucas D., Escudero B. et al., 2009) was its requirement for correct recombination of the immunoglobulin k light chain during B cell development. Thus, pol ¿ deficiency results in shorter, non-productive Vk-Jk junctions that lead to lymphocyte cell death at the transition from PreB to immature B cell stage. Unlike TdT, pol ¿ is expressed in many tissues (liver, kidney, lung, brain, testis), suggesting that it might participate in general NHEJ in vivo. This fact led us to search for additional functions where pol ¿ could be involved apart from the recombination of immunoglobulin genes. The pol ¿ deficient mouse (¿-/-) generated in our laboratory has shown reduced numbers of B lymphocytes, but also of neutrophils and platelets. Moreover, this mouse is hypersensitive to ionizing radiation. Strickingly, this mouse model has a phenotype of Summary 10 delayed aging, as shown, for example, by the extended lifespan and reduced deterioration of brain function with age. In the present work, we characterize in detail the function of pol ¿ in general NHEJ pathway, analyzing the effects of its efficiency under stressful conditions in hematopoietic as well as in non hematopoietic cells/tissues. We observed that pol ¿ deficient primary cells are sensitive to ionizing radiation and that ¿-/- MEFs enter senescence as a consecuence of DNA damage and genomic stability generated by stressful culture conditions. We have also characterized, in part, the role of this polymerase in the aging process in mice. Our results indicate that pol¿-/- aging livers show reduced apoptosis and polyploidy, both related to liver aging. These data are in agreement with the notion that the lack of this polymerase can cause a delay in aging. Moreover, reactive oxygen species (ROS) acummulation, which is a key factor contributing to organism deterioration, was also found to be diminished in ¿-/- thymus and spleen. To further investigate the function of pol ¿ in DSBs repair, we generated a double knockout mouse model which combines deficiencies for pol ¿ and p53. Surprisingly, ¿- /-p53-/- mice shows reduced incidence of lymphoma compared to p53-/- mice, but increased incidence of sarcoma, suggesting that pol ¿ plays a role in non lymphocytic cells. ¿-/-p53-/- mice have a significantly shorter lifespan than p53-/- mice. We propose that this phenotype is due to the higher sarcoma incidence. The cooperation between p53 and pol ¿ in mesodermic tissues is strengthned by our in vitro results that indicate that MEFs deficient in both ¿-/- and p53-/- are more chromosomically unstable than p53-/- MEFs. All together, these data demonstrate that pol ¿ participates in general NHEJ pathway: 1) Its abscence affects the hematopoietic system and non hematopoietic tissues, as shown by the higher chromosome instability and/or ¿H2AX levels in médula ósea, spleen, liver and MEFs ¿-/- under genotoxic stress. This role of pol ¿ is supported by the fact that the absence of this polymerase promotes sarcomas, conjunctive tissue-derived tumors, in p53-/- genetic background. 2) Pol ¿ deficiency delays aging, as shown by the increased lifespan and by several ¿younger¿ ¿-/- tissues compared to controls. We propose that the lack of pol ¿ makes the NHEJ pathway less error-prone (probably because pol ¿ is replaced by other polymerases without transfer terminal activivity (i.e. pol ¿) but slightly slower and less efficient. In situations of DNA damage, in a pol¿ null context, HR repairs some of the breaks with high fidelity and the cells that cannot efficiently repair the damage, are eliminated by apoptosis. We propose that by increasing HR and decreasing mutagenic NHEJ, ¿-/-mice accumulate fewer DNA mutations throughout life, promoting longevity.