INO80, a member of the multi-subunit SWI2/SNF2 superfamily, is involved in transcription regulation, DNA repair and replication. Not much is known about its substrate specificity and remodeling mechanism or how it differs in comparison to SWI/SNF or ISWI. Site-directed mapping of histone-DNA contacts showed that INO80 generally remodels mononucleosomes by moving them to the center of DNA. The length of extranucleosomal DNA was found to play an important role in nucleosome binding as well as remodeling by INO80 much like ISW2 and ISW1a. INO80 preferentially binds to nucleosomes containing >20bp of extranucleosomal DNA. Similarly, INO80 remodeling of mononucleosomes with different lengths of extranucleosomal DNA showed that at least 33bp of extranucleosomal DNA on one side of the nucleosome was required for initiation of remodeling. These data suggest that INO80 behaves much like ISW2 and ISW1a complexes based on their requirement for extranucleosomal DNA. INO80 does not unravel or displace nucleosomes like SWI/SNF. There are several key aspects of how INO80 interacts with and remodels nucleosomes that are quite distinct from SWI/SNF, ISW2, and ISW1a. Previously SWI/SNF and ISW2 were shown to initiate nucleosome movement by translocating along nucleosomal DNA two helical turns from the dyad axis. Nucleosome movement by INO80 instead requires translocation by the complex along nucleosomal DNA near the entry/exit site at the dimer-tetramer interface. Sliding interference of INO80 by the presence of nicks indicated that torsional strain at the site of translocation is required for nucleosome mobilization by INO80. Hydroxyl radical footprinting of the INO80-nucleosome complex shows found that INO80 interactioninteracts with extranucleosomal DNA at, the entry-exit site and to lesser extent at the dyad axis, but it lacks the protection found indoes not contact 2 helical turns from the dyad like ISW2 and SWI/SNF at two helical turns from the dyad axis as determined by photoaffinity cross-linking studies. The catalytic subunit (Ino80) rather than being found associated 2 helical turns from the dyad, was bound to extranucleosomal DNA and nucleosomal DNA near the entry-exit site. Other subunits (Arp8p, Arp5p and Nhp10) were also found to be contacting both nucleosomal and extranucleosomal DNA. Site-specific histone cross-linking studies revealed that Ino80, Arp5 and Arp4 interact extensively with the histone dimer of the nucleosome in comparison to H3-H4 tetramer. Although N-terminal histone tails are often important for chromatin remodeling, INO80 shows no requirement of histone tails for its nucleosome binding and mobilizing activities. The deviation of INO80 from the canonical model of how ATP-dependent remodelers interact and mobilize nucleosome is apparently due to its unique role as a member of the remodeling complexes that promote the exchange of H2A/H2B dimer from core nucleosome particle.
| Identifer | oai:union.ndltd.org:siu.edu/oai:opensiuc.lib.siu.edu:dissertations-1235 |
| Date | 01 December 2010 |
| Creators | Udugama, Maheshi Imalka |
| Publisher | OpenSIUC |
| Source Sets | Southern Illinois University Carbondale |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations |
Page generated in 0.0017 seconds