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Protein-DNA interactions and chromosome condensation

University of British Columbia

The study of protein-DNA interactions has become a major focus of biochemical research since the realization that they are at the heart of such basic cellular processes as transcription, replication, recombination, and chromosome condensation. In vitro, many different techniques are available to study several aspects of protein-DNA interactions, however, their often transient nature can impede adequate analyses. In vivo, the 2 m of DNA that must be densely packed into a nucleus 5 μm in diameter, requires a complex system of interactions between proteins and DNA that are much more challenging to study. Ultraviolet laser crosslinking of proteins to DNA is a potentially powerful tool for studying their interactions in vitro and in vivo. Described in this thesis are procedures to detect and isolate covalently crosslinked protein-DNA complexes, both in vitro and in vivo. Analysis of crosslinked protein-DNA complexes by SDS-PAGE furnishes the approximate molecular weight of the crosslinked protein. Analysis of the DNA sequence bound to protein was not possible, as the DNA was determined to be excessively damaged, and thus unable to be replicated by transformation into E. coli, or amplified by the polymerase chain reaction. To highlight one of its many practical applications, in vivo UV laser crosslinking was used together with in situ approaches, to demonstrate that the histone H3 aminoterminal tail is bound to DNA during interphase, but upon histone H3 phosphorylation and chromosome condensation at mitosis, the tail becomes unbound. This is supported by in situ studies, which showed that the histone H3 tail is more accessible to antibodies during mitosis than at interphase. These observations correlate well with the mitotic phosphorylation of histone H3. In addition, polyamines were shown to associate with mitotic chromosomes to a greater extent than with decondensed chromatin. The evidence presented here suggests a new model for the role of the H3 tail at chromosome condensation. During interphase, the H3 tail is unphosphorylated and bound to DNA , and the chromatin is decondensed. At mitosis, the H3 tail becomes phosphorylated and is released from DNA, chromosomes condense, and polyamines become heavily associated with mitotic chromosomes, perhaps aiding in their packaging.

Thesis/Dissertation

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2009-06-02

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http://hdl.handle.net/2429/8585

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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