Interplay between mismatch repair and chromatin assembly - PubMed (original) (raw)

Interplay between mismatch repair and chromatin assembly

Barbara Schöpf et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Single strand nicks and gaps in DNA have been reported to increase the efficiency of nucleosome loading mediated by chromatin assembly factor 1 (CAF-1). However, on mismatch-containing substrates, these strand discontinuities are utilized by the mismatch repair (MMR) system as loading sites for exonuclease 1, at which degradation of the error-containing strand commences. Because packaging of DNA into chromatin might inhibit MMR, we were interested to learn whether chromatin assembly is differentially regulated on heteroduplex and homoduplex substrates. We now show that the presence of a mismatch in a nicked plasmid substrate delays nucleosome loading in human cell extracts. Our data also suggest that, once the mismatch is removed, repair of the single-stranded gap is accompanied by efficient nucleosome loading. We postulated that the balance between MMR and chromatin assembly might be governed by proliferating cell nuclear antigen (PCNA), the processivity factor of replicative DNA polymerases, which is loaded at DNA termini and which interacts with the MSH6 subunit of the mismatch recognition factor MutSα, as well as with CAF-1. We now show that this regulation might be more complex; MutSα and CAF-1 interact not only with PCNA, but also with each other. In vivo this interaction increases during S-phase and may be controlled by the phosphorylation status of the p150 subunit of CAF-1.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Ongoing MMR delays chromatin assembly. (A) Kinetics of supercoiling of nicked mismatch-containing (G/Tnicked) DNA substrates incubated with nuclear extracts of human MutSα-deficient LoVo cells, supplemented where indicated with recombinant MutSα. The figure shows a UV shadowing of an agarose gel poststained with EtBr (Upper) and its autoradiograph (32P, Lower). Migration positions of the open/nicked circular (Ir/II) and supercoiled DNA (I) isoforms are indicated. (B) Quantitation of three independent G/Tnicked supercoiling experiments (A shows a representative example), as a ratio of the sum of all covalently closed topoisomers (cc) versus total DNA. (C) Supercoiling of homoduplex (A/T, A/Tnicked) and heteroduplex (G/T, G/Tnicked) substrates incubated with nuclear extracts of human MutSα-deficient LoVo cells, supplemented where indicated with recombinant MutSα. Data from three independent experiments are shown. The error bars represent the standard deviation from the mean. (D) Quantitation of supercoiling assays of A/Tnicked and G/Tnicked substrates after incubation with nuclear extracts of human MutSα-deficient LoVo cells, supplemented where indicated with recombinant MutSα—either wild type, or its variants KR (ATPase mutant), FA (DNA binding mutant), or C1 (PCNA interaction mutant). Data from three independent experiments were analyzed. The error bars represent the standard deviation from the mean.

Fig. 2.

Fig. 2.

Effect of CAF-1 on the efficiency of chromatin assembly and MMR. (A) Representative supercoiling/repair assay of A/Tnicked and G/Tnicked substrates in nuclear extracts of LoVo cells supplemented with MutSα and/or with recombinant CAF-1 as indicated. (B) MMR efficiency in the extracts was estimated by recovering the G/Tnicked substrates following incubation with the extracts and digestion with _Acl_I. As shown, addition of CAF-1 to the assay did not alter MMR efficiency. (C) After 10 min preincubation (+) of the A/Tnicked and G/Tnicked substrates with LoVo extracts supplemented (+) or not (−) with MutSα. In the control experiments (−), the extract was incubated under identical conditions, and the substrates were added after 10 min together with the MutSα. The extent of supercoiling was examined 15 min later. Data from three independent experiments were analyzed. The error bars represent the standard deviation from the mean. (D) MMR assay of the G/Tnicked substrate preincubated with (+) or without (−) MutSα as indicated.

Fig. 3.

Fig. 3.

MutSα interacts directly with the p150 subunit of CAF-1. (A) Anti-MSH6 coimmunoprecipitations were analyzed by Western blotting for CAF-1. Ten percent HeLa nuclear extracts served as the input control. (B) Extracts of 293 cells stably expressing FLAG-CAF-1 p150 were incubated with anti-FLAG beads. Elution was done using FLAG peptides. The control was 293 cells expressing FLAG only (empty). The input control was 0.5% of eluted material. Input fraction (I), Unbound material (U), Eluate (E). Asterisk marks leftover signal of previous p150 blot. (C) Far Western blot showing a direct interaction between MutSα and CAF-1. The CAF-1 trimer was separated by SDS-PAGE, transferred onto a membrane, incubated with MutSα and hybridized with an anti-MSH6 antibody. BSA was used as negative control. (D) Schematic representation of human MSH6. The PWWP (red), DNA-binding (orange), and ATPase (light blue) domains are indicated. The clamp region (dark green) is located within the lever domain (light green) that follows the connector domain (yellow). The GST-MSH6 fusion fragments that interacted with purified p150 in a GST pull-down experiment are shown in red, fragments that did not interact are in blue. (E) Schematic representation of the p150 subunit of human CAF-1. MIR (MOD1-interacting region), PEST (yellow), as well as KER (red), and ED (green) histone interacting domains are shown. The PCNA-binding motif (orange) as well as the p60-interacting region (light blue) are also indicated. The C-terminal half of p150 is needed for replication-coupled assembly. The GST-p150 fragments that interacted with purified MutSα in a GST pull-down experiment are indicated in red, those that did not interact are shown in blue.

Fig. 4.

Fig. 4.

Interactions between MutSα, CAF-1, and PCNA are differentially affected by treatment with DNA damaging agents. (A) Cell extracts from different cell cycle stages (

Fig. S4_A_

) were immunoprecipitated with an anti-MSH6 antibody and analyzed by Western blotting. (B) U2OS cells were pretreated with _O_6-benzylguanine to inhibit methylguanine methyl transferase before treatment with 10 μM MNNG. Anti-MSH6 immunoprecipitates were analyzed for MSH6, CAF-1 p150, or PCNA. (C) Lambda phosphatase treated or untreated purified, recombinant MutSα and CAF-1 p150 were used in immunoprecipitations with an anti-MSH6 antibody. The prefix p indicates polypeptides endogenously phosphorylated in Sf9 cells.

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