USP37 deubiquitinates Cdt1 and contributes to regulate DNA replication

Abstract

DNA replication control is a key process in maintaining genomic integrity. Monitoring DNA replication initiation is particularly important as it needs to be coordinated with other cellular events and should occur only once per cell cycle. Crucial players in the initiation of DNA replication are the ORC protein complex, marking the origin of replication, and the Cdt1 and Cdc6 proteins, that license these origins to replicate by recruiting the MCM2-7 helicase. To accurately achieve its functions, Cdt1 is tightly regulated. Cdt1 levels are high from metaphase and during G1 and low in S/G2 phases of the cell cycle. This control is achieved, among other processes, by ubiquitination and proteasomal degradation. In an overexpression screen for Cdt1 deubiquitinating enzymes, we isolated USP37, to date the first ubiquitin hydrolase controlling Cdt1. USP37 overexpression stabilizes Cdt1, most likely a phosphorylated form of the protein. In contrast, USP37 knock down destabilizes Cdt1, predominantly during G1 and G1/S phases of the cell cycle. USP37 interacts with Cdt1 and is able to de-ubiquitinate Cdt1 in vivo and, USP37 is able to regulate the loading of MCM complexes onto the chromatin. In addition, downregulation of USP37 reduces DNA replication fork speed. Taken together, here we show that the deubiquitinase USP37 plays an important role in the regulation of DNA replication. Whether this is achieved via Cdt1, a central protein in this process, which we have shown to be stabilized by USP37, or via additional factors, remains to be tested.

Keywords: DNA damage; DNA damage response; Initiation of DNA replication; Proteasome; Protein degradation; Ubiquitin hydrolase.

Figure 1

USP37 controls Cdt1 levels. (A) 293T cells were transfected with an empty vector (EV) or expression plasmids for the indicated DUBs and when indicated treated with UV light (40 J/m2) and collected 30 min later. Cell lysates were analyzed by Western blot with the indicated antibodies. (B) 293T cells transfected with EV or Flag‐USP37 were lysed for analysis of mRNA levels by real‐time PCR or by Western blot with the indicated antibodies. The graph shows the average Cdt1 mRNA levels of 3 independent experiments. (C) 293T cells were transfected with EV control or Flag‐USP37. Then, cells were left untreated or treated with 40 J/m2 UV light before collecting them at the indicated times (min). Extracts were analyzed by Western blot. (D) 293T cells transfected with Flag‐USP37 were incubated with thymidine for 24 h and released in fresh medium for 4 h. Then cells were left untreated or treated with UV light (40 J/m2) and collected 30 min post‐treatment before Western blot (left) or by flow cytometry (propidium iodide) analysis. (E) 293T cells were transfected with Flag‐USP37 or empty vectors and were labeled with BrdU (30 min) for FACS analysis. The percentage of BrdU positive cells is indicated. (F) U2OS cells were transfected with the indicated siRNA oligos and left untreated or UV irradiated (40 J/m2) before collection at the indicated times and analysis by Western blot. Quantification of Cdt1 levels compared to the loading control β‐actin is shown at the bottom. In this quantification, Cdt1 levels in all samples were compared to those in siLuc cells without damage (set as 1, upper row). Also, samples of USP37 depleted cells were compared to the undamaged control (set to 1, bottom row).

Figure 2

USP37 stabilizes a phosphorylated form of Cdt1. (A) 293T cells transfected with empty or Flag‐USP37 vector were lysed and loaded three times in the same gel for parallel Western blot analysis with three different Cdt1 antibodies. The anti‐Cdt1 immunoblot intensities were different and comparable exposures in each case are shown. (B) 293T cells were transfected with control or USP37 siRNA oligos and left untreated or treated with MG132 for 6 h when indicated. Cell lysates were analyzed with the indicated antibodies. (C) The same as (B), but using U2OS cells. (D) Endogenous Cdt1 was immunoprecipitated with the anti‐Cdt1 antibody from lysates of 293T cells overexpressing USP37. Input and immunoprecipitates were analyzed by Western blot with the indicated antibodies. Two different exposures of the same blot are shown. (E) 293T cells transfected with empty or Flag‐USP37 overexpression vectors were lysed. Extracts were treated or not with lambda phosphatase for 60 min prior analysis by Western blotting with the indicated antibodies. (F) 293T cells overexpressing Flag‐HA‐USP37 were lysed and anti‐Cdt1 immunoprecipitations were carried out. The indicated immunoprecipitates were incubated with lambda phosphatase for 60 min before analysis by Western blot with the indicated antibodies.

Figure 3

USP37 interacts with and deubiquitinates Cdt1. (A) 293T cells transfected with an empty vector or Flag‐USP37 were lysed and immunoprecipitations were carried out with anti‐Flag beads. Immunoprecipitates were analyzed by Western blot with anti‐Cdt1 and anti‐USP37 antibodies. (B) As in (A), but when indicated, cells were UV irradiated (40 J/m2, 1 h). (C) 293T cells were transfected with a plasmid expressing Strep‐Cdt1 together with control or His‐Ubiquitin expression plasmids, and wild type or catalytic inactive Flag‐USP37. 20 h after transfection, cells were incubated with MG132 for 16 h before lysis under denaturing conditions. Western blot analysis of input and His‐pull downs were carried out with the indicated antibodies.

Figure 4

USP37 controls Cdt1 levels during G1 and G1/S. (A) U2OS were left asynchronous or synchronized using different protocols as described in Materials and Methods. When indicated, 30 min before collecting, cells were treated with 40 J/m2 UV light. Then samples were collected for propidium iodide analysis by flow cytometry (right) or Western blot with the indicated antibodies (left). (B) U2OS were transfected with control or USP37 siRNA oligos and synchronized at the same time as described. A fraction of cells was lysed for Western blot analysis with the indicated antibodies (left) and another fraction of cells was collected for flow cytometry analysis after propidium iodide staining (right).

Figure 5

USP37 controls replication fork speed and impacts on MCM loading. U2OS cells were left untreated or transfected with control or USP37 siRNA oligos (A) or 293T were left untreated or transfected with an empty or Flag‐USP37 expressing plasmid (B). 48 h after transfections cells were labeled with nucleotide analogues as described in materials and methods. Cells were then collected for DNA fiber analysis, for Western blotting with the indicated antibodies and for flow cytometry analysis (BrdU/PI). Depicted is the replication fork speed and the percentage of origin firing from two different experiments. Horizontal lines represent the median of relative fork speed distribution. Western blots and flow cytometry graphs are shown from a representative experiment. (C) 293T cells transfected with an empty vector or Flag‐USP37 expressing plasmid were synchronized by thymidine block and collected 4 h after thymidine release for flow cytometry analysis or for biochemical fractionation and Western blot analysis with the indicated antibodies. S1 + S2 represent soluble proteins, P2 the chromatin fraction. Quantification of the MCM7 levels in the chromatin fraction compared to the levels of histone H3 is represented. In the first row the MCM7 levels are compared to EV without damage, the second row compares −/+ Flag‐USP37 expression in UV‐treated cells. (D) Model summarizing previously known data and the new data from this article. USP37 deubiquitinates a phosphorylated, unstable form of Cdt1. USP37 also delays replication fork speed by a mechanism likely independent of Cdt1.