Structure and function of chromatin

Within the period 1978-1990 the Department’s research activity developed in several directions. ● Structure of 30 nm chromatin fiber: the role of histone H1/H5/ and their localization within the fiber (the first demonstration that histone H5 is internally located; an original triple-helix model was suggested) ● Thermal denaturation of chromatin as an approach for studying of chromatin structure (demonstration of nucleosome sliding upon melting of H1-depleted chromatin)

● Transcribed chromatin: methods for isolation, non-histone complement, acetylation of histones (some of the earlier data for hyperacetylation of active chromatin) ● Satellite chromatin as an example of firmly repressed chromatin: separation, isolation of pericentromeric chromatin (the first evidence that histones of satellite chromatin are not acetylated) ● Photocrosslinking protein to DNA in vivo by using nano- and picosecond UV laser (the first method for laser crosslinking in vivo) ● Chromatin structure of ribosomal genes as a function of their expression (the first evidence that the active ribosomal genes do not represent a naked  DNA but contain all histones; core histones associated with the actively transcribed gene copies are hyperacetylated, those bound to nucleosome-organised genes are not.

            After 1990 the research was switched to the non-histone chromosomal protein HMGB1, serving the function of “architectural” factor of chromatin. This protein is studied in three aspects: ● DNA binding properties with an emphasis on its ability to bind DNA damaged by the antitumor drug cisplatin, ● Post-synthetic modifications of HMGB1 and ● The role  of the acidic C-terminal domain for the properties of the protein. The main results so far reported are (i) HMGB1 binds preferentially to UV-damaged DNA; (ii) Isolation, purification, molecular characterization and DNA binding properties of in vivo acetylated HMGB1 ; (iv) identification of the enzyme that acetylated the protein in vitro: this is the histone transacetylase CBP which also modifies Lys2.

Another research area is the regulation of DNA unwinding during replication. Basic conclusions ● The replicative helicase can unwind part of DNA in the absence of DNA synthesis in vivo, suggesting the existence of checkpoint mechanism for regulation of DNA unwinding ● A specific checkpoint complex was identified, interacting directly with e helicase during  replication fork progression and when the fork is stalled.