Graduate Thesis Or Dissertation

 

Interaction of linker proteins, H1 and HMG1, with nucleosome reconstituted on positioning sequences Public Deposited

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/79408047j

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  • The aim of this research was to gain more understanding on how a linker histone (LH), H1⁰, and high mobility group protein, HMG1, interact with nucleosome. To determine the location of linker proteins, three specific positioning sequences were used to reconstitute core nucleosomes and the binding of H1 and HMG1 to them was studied by nuclease protection. As a first step, a report of asymmetric positioning of LH on mono-nucleosome reconstituted with a cloned fragment containing the 5S rDNA from X. borealis was reinvestigated. It was found that this sequence has two major core positions and that the previously reported 5 15 by protection by LH is an artifact of MNase digestion. On the other hand, it seems clear that LH protects ~20bp linker DNA on only one side of 3-151 core nucleosome. Using two alternative DNA sequences, a 243-bp fragment containing the L. variegates 5S rDNA and a 235-bp fragment derived from the clone called pGUB, it was found that LH protects DNA on only one side of the core particles, and more importantly, the asymmetric, one-side protection of linker DNA is sequence dependent. Cross linking experiments additionally demonstrated that the binding of linker proteins to the core nucleosome causes neither a change in nucleosome structure nor sliding of the histone octamer along the DNA sequences. The possible contributions of octamer histone tails to the positioning of the LH was also examined using trypsinized histone octamers for reconstitution. The results showed that the binding of LH onto core nucleosome is slightly facilitated by the presence of histone tails but the absence of histone tails did not change the protection by LH, indicating that the core histone tails are not involved in locating LH on core nucleosome. Finally, the position of HMG1 on nucleosomes was studied and the results showed that HMG1 protects linker DNA on one side of the core particle. Importantly, the linker DNA site protected b y HMG1 was located on the side opposite to that already found to b e protected by linker histone binding.
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