Localization of the Drosophila Rad9 Protein to the Nuclear Membrane Is Regulated by the C-Terminal Region and Is Affected in the Meiotic Checkpoint

Rad9, Rad1, and Hus1 (9-1-1) are part of the DNA integrity checkpoint control system. It was shown previously that the C-terminal end of the human Rad9 protein, which contains a nuclear localization sequence (NLS) nearby, is critical for the nuclear transport of Rad1 and Hus1. In this study, we show that in Drosophila, Hus1 is found in the cytoplasm, Rad1 is found throughout the entire cell and that Rad9 (DmRad9) is a nuclear protein. More specifically, DmRad9 exists in two alternatively spliced forms, DmRad9A and DmRad9B, where DmRad9B is localized at the cell nucleus, and DmRad9A is found on the nuclear membrane both in Drosophila tissues and also when expressed in mammalian cells. Whereas both alternatively spliced forms of DmRad9 contain a common NLS near the C terminus, the 32 C-terminal residues of DmRad9A, specific to this alternative splice form, are required for targeting the protein to the nuclear membrane. We further show that activation of a meiotic checkpoint by a DNA repair gene defect but not defects in the anchoring of meiotic chromosomes to the oocyte nuclear envelope upon ectopic expression of non-phosphorylatable Barrier to Autointegration Factor (BAF) dramatically affects DmRad9A localization. Thus, by studying the localization pattern of DmRad9, our study reveals that the DmRad9A C-terminal region targets the protein to the nuclear membrane, where it might play a role in response to the activation of the meiotic checkpoint

The human checkpoint sensor Rad9–Rad1–Hus1 interacts with and stimulates DNA repair enzyme TDG glycosylase

Human (h) DNA repair enzyme thymine DNA glycosylase (hTDG) is a key DNA glycosylase in the base excision repair (BER) pathway that repairs deaminated cytosines and 5-methyl-cytosines. The cell cycle checkpoint protein Rad9–Rad1–Hus1 (the 9-1-1 complex) is the surveillance machinery involved in the preservation of genome stability. In this study, we show that hTDG interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. The hHus1 interacting domain is mapped to residues 67–110 of hTDG, and Val74 of hTDG plays an important role in the TDG–Hus1 interaction. In contrast to the core domain of hTDG (residues 110–308), hTDG(67–308) removes U and T from U/G and T/G mispairs, respectively, with similar rates as native hTDG. Human TDG activity is significantly stimulated by hHus1, hRad1, hRad9 separately, and by the 9-1-1 complex. Interestingly, the interaction between hRad9 and hTDG, as detected by co-immunoprecipitation (Co-IP), is enhanced following N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment. A significant fraction of the hTDG nuclear foci co-localize with hRad9 foci in cells treated with methylating agents. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of the BER

Gastrointestinal Hyperplasia with Altered Expression of DNA Polymerase β

Background: Altered expression of DNA polymerase β (Pol β) has been documented in a large percentage of human tumors. However, tumor prevalence or predisposition resulting from Pol β over-expression has not yet been evaluated in a mouse model. Methodology/Principal Findings: We have recently developed a novel transgenic mouse model that over-expresses Pol β. These mice present with an elevated incidence of spontaneous histologic lesions, including cataracts, hyperplasia of Brunner's gland and mucosal hyperplasia in the duodenum. In addition, osteogenic tumors in mice tails, such as osteoma and osteosarcoma were detected. This is the first report of elevated tumor incidence in a mouse model of Pol β over-expression. These findings prompted an evaluation of human gastrointestinal tumors with regard to Pol β expression. We observed elevated expression of Pol β in stomach adenomas and thyroid follicular carcinomas, but reduced Pol β expression in esophageal adenocarcinomas and squamous carcinomas. Conclusions/Significance: These data support the hypothesis that balanced and proficient base excision repair protein expression and base excision repair capacity is required for genome stability and protection from hyperplasia and tumor formation

Long Patch Base Excision Repair Proceeds via Coordinated Stimulation of the Multienzyme DNA Repair Complex*

Base excision repair, a major repair pathway in mammalian cells, is responsible for correcting DNA base damage and maintaining genomic integrity. Recent reports show that the Rad9-Rad1-Hus1 complex (9-1-1) stimulates enzymes proposed to perform a long patch-base excision repair sub-pathway (LP-BER), including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase β (pol β), flap endonuclease 1 (FEN1), and DNA ligase I (LigI). However, 9-1-1 was found to produce minimal stimulation of FEN1 and LigI in the context of a complete reconstitution of LP-BER. We show here that pol β is a robust stimulator of FEN1 and a moderate stimulator of LigI. Apparently, there is a maximum possible stimulation of these two proteins such that after responding to pol β or another protein in the repair complex, only a small additional response to 9-1-1 is allowed. The 9-1-1 sliding clamp structure must serve primarily to coordinate enzyme actions rather than enhancing rate. Significantly, stimulation by the polymerase involves interaction of primer terminus-bound pol β with FEN1 and LigI. This observation provides compelling evidence that the proposed LP-BER pathway is actually employed in cells. Moreover, this pathway has been proposed to function by sequential enzyme actions in a “hit and run” mechanism. Our results imply that this mechanism is still carried out, but in the context of a multienzyme complex that remains structurally intact during the repair process