Abstract
Double-strand breaks (DSBs) constitute the most deleterious form of DNA lesions that can lead to genome alterations and cell death, and the vast majority of DSBs arise pathologically in response to DNA damaging agents such as ionizing radiation (IR) and chemotherapeutic agents. Recent studies have implicated a role for the human MutS homologue hMSH5 in homologous recombination (HR)-mediated DSB repair and the DNA damage response. In the present study, we show that hMSH5 promotes HR-based DSB repair, and this property resides in the carboxyl-terminal portion of the protein. Our results demonstrate that DSB-triggered hMSH5 chromatin association peaks at the proximal regions of the DSB and decreases gradually with increased distance from the break. Furthermore, the DSB-triggered hMSH5 chromatin association is preceded by and relies on the assembly of hMRE11 and hRad51 at the proximal regions of the DSB. Lastly, the potential effects of hMSH5 non-synonymous variants (L85F, Y202C, V206F, R351G, L377F, and P786S) on HR and cell survival in response to DSB-inducing anticancer agents have been analyzed. These experiments show that the expression of hMSH5 variants elicits different survival responses to anticancer drugs cisplatin, bleomycin, doxorubicin and camptothecin. However, the effects of hMSH5 variants on survival responses to DSB-inducing agents are not directly correlated to their effects exerted on HR-mediated DSB repair, suggesting that the roles of hMSH5 variants in the processes of DNA damage response and repair are multifaceted.
Highlights
The MutS homologue hMSH5 is a member of the DNA mismatch repair (MMR) family of proteins [1,2,3]
Recombinant hMSH5 protein interacts with hMSH4–the only other MutS homologous protein possessing no apparent role in the process of MMR–to form a heterocomplex that can recognize several Holliday junction (HJ) intermediate structures resembling those arisen during recombinational Double-strand breaks (DSBs) repair [13]
Repair was first assessed by the homologous recombination (HR) reporter system 293TLa/ pMMR-IR3 that measures the frequency of recombination initiated by an I-SceI-induced DSB (Fig. 1A) [29]. 293TLa is an hMLH1 Tet-off cell line, in which the expression of hMLH1 can be repressed by doxycycline [34]
Summary
The MutS homologue hMSH5 is a member of the DNA mismatch repair (MMR) family of proteins [1,2,3]. Recombinant hMSH5 protein interacts with hMSH4–the only other MutS homologous protein possessing no apparent role in the process of MMR–to form a heterocomplex that can recognize several Holliday junction (HJ) intermediate structures resembling those arisen during recombinational DSB repair [13]. It is important to note that the expression patterns of hMSH5 and hMSH4 differ significantly in mitotic tissues – of which hMSH5 is broadly expressed in a variety of organs; in contrast, expression of hMSH4 is considerably limited [1,2,3,5,18,19] This observation suggests that hMSH5 may act independently of hMSH4 through networking with other proteins. The levels of protein expression in cells are often low (MOPED or the Model Organism Protein Expression Database), hMSH5 could undergo induction and become phosphorylated in cells treated with ionizing radiation (IR) or cisplatin [14,15,20], raising the possibility that this MutS homologue may play a role in the process of DSB repair
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