Fig 1: MSH2–MSH3 stimulates pol β DNA synthesis and enhances TNR flap size in the context of BER.(a) The schematic representation of the (GAA)20 or (CAG)20 substrates with a 32P-label on the 5′-end of the damaged strand. (b) The labelled bands correspond to the length of pol β extension products in the absence or presence of MSH2–MSH3. The (GAA)20 (b) or (CAG)20 (c) substrates (25 nM) were incubated at 37 °C with 25 nM APE1 and 10 nM pol β in the absence or presence of 100 nM MSH2–MSH3. The machinery present in each extension reaction is indicated above the gels; (+) is the presence of the component and (−) is the absence of the component. BSA substitutes for MSH2–MSH3 in reactions in lanes 5 and 10. The number of repeats added is indicated to the right of the gels. (d) A schematic illustration of the results from b and c. (e) A schematic diagram of an open-template substrate containing random DNA sequence 32P-labelled at the 5′-end of the primer. (f) DNA synthesis activity from purified MSH2–MSH3 proteins was measured by incubating the open-template substrate shown in e without (lane 1) or with 100–500 nM of MSH2–MSH3 (lanes 2–4). Lane 5 represents reaction mixtures with 5 U Pol I Klenow fragment as a positive control. An extension product is observed only in lane 5. (g–j) The effect of MSH2–MSH3 on the size of the displaced strand during active BER after APE1 cleavage and pol β extension. (g) Schematic diagram of the (GAA)20 or (CAG)20 substrate illustrating the THF site and the position of 32P-labelling at the 3′-end of the damaged strand and the APE1 site (black arrow and star). Pol β DNA synthesis on the substrate generates displaced flaps, whose size is measured by FEN1 flap cleavage. (h,i) FEN1 cleavage products after pol β DNA synthesis of the (GAA)20 (h) or (CAG)20 (i) substrate. The concentration of FEN1 in the reaction is 10 nM, and all other reagents are the same as in b and c. Black dots and numbers to the right of the gel indicate the sizes of the FEN1 cleavage products. (j,k) Schematic representations of the results of h and i. Experiments were done in triplicate. rpts, repeats.
Fig 2: MSH2–MSH3 and pol β co-localize in cells to form physical complexes that increase with treatment by DNA-damaging agents.Co-immunoprecipitation (IP) and immunoblotting (IB) of MSH2 or pol β in lymphoblast cell extracts derived from a normal individual (GM02152) were either untreated (a) or treated with 0.5 mM K2CrO4 (b) or 10 mM KBrO3 (c) for 2 h, respectively. (d) Same as a except for LoVo cells that lack MSH2. Cell lysates were subjected to co-IP and immunoblotting for pol β and MSH2, as described in the Methods section. Lanes 1 and 2 are the purified pol β or MSH2 proteins, as indicated, detected with their respective antibodies. Lane 3 corresponds to cell lysates without treatment as an ‘input' control. Lane 4 is the cell lysates immunoprecipitated with a rabbit IgG alone; lanes 5 and 6 are cell lysates immunoprecipitated with an anti-pol β antibody and an anti-MSH2 antibody, respectively. (e) Same as a–d, but for purified proteins. Purified pol β and MSH2 proteins were employed as a molecular weight marker (lanes 1 and 2). (f,g) Co-localization of MSH2 and pol β in normal lymphoblasts (GM02152) (f) and LoVo cells (g) untreated or treated with oxidative DNA-damaging agents, potassium bromate or potassium chromate, as indicated. Representative images are illustrated. MSH2 (red), pol β (green) or merged images are indicated. Zeiss pixel analysis results are illustrated to the right side of each panel. The x and y axes represent red and green staining intensity, respectively. The white bars represent the quadrant thresholds below which pixels were deemed to contain only red or only green staining. The number 3 is the quadrant in which pixels contain both red and green intensities. 4,6-Diamidino-2-phenylindole (blue) is a stain for nuclear DNA; red is the anti-MSH2 staining; green is anti-pol β staining. (g) Same as f for untreated LoVo cells. All experiments were carried out in triplicate. All scale bars, 10μm.
Fig 3: DNA repair genes are dysregulated in HD stem cell lines, as well as patient brains, but show differing directions of effect at the transcript and protein level. Gene expression of DNA repair genes, (A) PMS2, (B) FAN1 and (C) MSH3, was elevated at longer CAG lengths in human pluripotent stems cells (hPSCs, left), but relative protein levels are decreased in HD patient brains compared with matched controls (middle and right). Asterisks indicate P < 0.05.
Fig 4: MSH2–MSH3 suppresses TNR deletion and promotes repeat expansion during BER.The repair products were pulled down with avidin beads after the termination of the BER reaction. The repaired strands were subsequently separated from their biotinylated template strands by incubating with NaOH. The separated repaired strands were subsequently amplified using 5′-FAM-labelled primers and resolved by high-resolution capillary electrophoresis to define their length. (a) A schematic diagram of (GAA)20 or (CAG)20 substrate containing an abasic lesion. (b) A schematic diagram of the PCR amplification methodology and electrophoretic separation of the BER products. (c,d) GeneMapper scans of the ligated and amplified (GAA)20 (c) or (CAG)20 (d) products in the absence and presence of MSH2–MSH3. The top panel represents the size of the (GAA)20 or (CAG)20 templates without any damage or BER machinery added. The middle and lower panels illustrate DNA fragment analysis of the templates in a reconstituted with the BER machinery in the absence (middle) and presence (lower) of MSH2–MSH3. Key TNR sizes are indicated with black arrows. The red peaks in the scans indicate size standards, whose size is indicated below the scans. All experiments were performed in at least triplicate. rpt, repeat.
Fig 5: Model for a ‘toxic oxidation' cycle by MMR–BER crosstalk.MSH2–MSH3 promotes TNR expansion via suppression of repeat deletion during BER. Oxidative stress induces an oxidized DNA base in a TNR tract such as 8-oxoG. OGG1 removes the 8-oxoG and leaves an abasic site that is subsequently 5′-incised by APE1. The pol β·MSH2–MSH3 complex loads onto DNA at the APE1 incision site, and promotes DNA synthesis and flap formation. MSH2–MSH3 inhibits FEN1 removal of the flap. However, MSH2–MSH3 interaction with the loop reorients MSH2–MSH3 and allows flap re-alignment on the damaged strand to generate a new and shorter flap suitable for FEN1 cleavage and ligation. Incorporation of the loop by an endonuclease results in expansion. In the absence of MSH2–MSH3 (−MSH2–MSH3), pol β opens the template to generate single-strand DNA loop structure, and deletion of the template strand occurs after endonuclease excision. Thus MSH2–MSH3 suppresses deletion and promotes expansion.
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