Effects Of Homologous Recombination Defects On The Formation Of MN-γH2AX(+)

[Background]DNA damage, which occurs frequently and in diverse forms, is one of the main threats to cell survival, cell division and cellular functions. DNA damage can be caused by endogenous factors and environmental factors. In the long-term evolution process, all kinds of organisms acquired different types of repair ways that are used to repair the diversity of the DNA damage. As the most serious forms of DNA damage, double-strand breaks (DSBs) can be repaired via non-homologous end joining (NHEJ) or homologous recombination repair. Homologous recombination (HR) occurs between homologous sequences. HR is a multistep process that requires different proteins at different steps,such as BRCA1 and CtIP in early stage, RPA1 and RAD51 in middle stage and TOPI and MUS81 in late stage.Formation of micronuclei (MN) serves as an indicator of genomic instability and exposure to genotoxicants. They can derive from lagging chromosomes during mitosis or from broken chromosomes. Terradas and our laboratory reported a new type of micronuclei in previous studies. This kind of micronuclei are uniformly marked by H2AX phosphorylation, thus are called the MN-γ-H2AX (+). Their spontaneous frequency varies among cell lines and can account for 1/5 to 1/3 of the total number of micronuclei. Previous studies carried out in our laboratory showed that MN-γ-H2AX (+) can be preferentially induced by replication and oxidative stress. MN-γ-H2AX (+) frequency can be increased 2-3 times when cells are treated with hydroxyurea or aphidicolin for 24 hours. It was also shown that MN-γ-H2AX (+) are produced mainly in S phase. In addition, our laboratory showed that oxidative stress can also preferentially MN-γ-H2AX (+). Interestingly, the MN induced by replication stress can be partially reduced by antioxidant, suggesting that oxidative stress mediates a portion of the MN induced by replication stress. Because HR is critical in the cellular response to replication stress, especially collapsed replication forks, it is expected that defects in HR will lead to increased formation of MNγy-H2AX (+). In this current study we therefore depleted various components in HR pathway by RNA interference and examined their effects on the formation of MN-γ-H2AX(+). We found that the homologous recombination defect can effectively increase micronuclei frequency. However, it appears that depletion of the different members of the HR pathway have different consequences in terms of formation of MN-γ-H2AX (+). In addition, we also found that change of micronuclei frequency is associated with the change in cell cycle distribution and ROS level.[Objective]The purpose of our study is to further clarify the mechanism by which MN-γ-H2AX(+) forms, to determine the role of homologous recombination in reduce formation of micronuclei and to explore the feasibility of micronuclei as an indicator of defective homologous recombination repair.[Materials and methods]1. U2OS cells were treated by HU and the changes of MN was detected by Immuno fluorescence.2. U2OS cells were treated by siRNA to deplete various members of HR pathway,and the changes of MN was detected by Immunofluorescence.3. U2OS cells were treated by siRNA to deplete various members of NHEJ pathway,and the changes of MN was detected by Immunofluorescence.4. Flow cytometry was used to determine the level of ROS in U2OS cells in which genes involved in HR and NHEJ were depleted by RNAi.5. Flow cytometry was used to examine cell cycle distribution in U2OS cells under various conditions.[Results]1. Stall of replication forks, induced by hydroxyurea for two hours, had little effect on the occurrence of micronuclei, while collapse of replication forks, induced by hydroxyurea for 24 hours, caused preferential induction of MN-γ-H2AX (+).2. CtIP depletion had no effect on the incidence of MN-γ-H2AX (+).3. RAD51 depletion or inhibition specifically induced MN-γ-H2AX (+).4. Depletion of either TOPI or MUS81 specifically induced MN-γ-H2AX (+).5. LIG4 depletion increased the frequency of MN.6. ROS were induced by depletion of some of the members in HR and NHEJ.7. Cell cycle distribution was significantly altered by depletion of RAD51 or TOP1, but not by CtIP.[Conclusions]Depletion or inhibition of certain members in the HR pathway may preferentially induce MN-γ-H2AX (+).The extent to which MN-γ-H2AX(+) are induced appears to depend on the stage in which the HR member functions. Increased ROS caused by depletion of the HR members partially contribute to the increased frequency of MN-γ-H2AX (+). Formation of MN-γ-H2AX(+) is also associated with alteration in cell cycle distribution. MN formation is less affected by the depletion of LIG4.