Figure 8: MSH2 is a critical linker between TRIM29 and DNA MMR proteins.

(a) Effect of MSH2 knockdown on chromatin association of endogenous TRIM29 in HeLa S3 cells. HeLa S3 cells were transfected with siControl or siMSH2 and incubated for 48 h. Total proteins (10 μg) from cytoplasm, nucleoplasm and chromatin fractions were subjected to SDS–PAGE followed by western blotting. (b) Effect of MSH2 knockdown on chromatin association of FLAG-tagged TRIM29 in HeLa S3 cells. HeLa S3 cells stably expressing FLAG-tagged TRIM29 were transfected with siControl or siMSH2 and incubated for 48 h. Total proteins (10 μg) from the cytoplasm, nucleoplasm and chromatin fractions were subjected to SDS–PAGE followed by western blotting. (c) Silver staining of anti-FLAG immunoprecipitates from nuclear extracts of MSH2-depleted cells expressing FLAG-tagged TRIM29 on 4%–20% gradient SDS–PAGE gel. (d) Interaction of FLAG-TRIM29 with ATM. HeLa S3 cells stably expressing FLAG-tagged TRIM29 were transfected with siControl or siMSH2. After 48 h, nuclear extracts were prepared from each cell line. Anti-FLAG immunoprecipitates were prepared from nuclear extracts and analysed by western blotting. (e) Model for TRIM29 function in activation of DNA damage signalling. TRIM29 binds to modified histone tails and functions as a molecular scaffold with DNA MMR proteins. These interactions are responsible for efficient activation of DDR. The assembly of TRIM29 and DNA repair proteins into chromatin is dependent on pre-existing H3K36me2, H4K16Ac and H4K20me2.