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Mechanisms of transcription & replication through chromatin

by
Han-Wen Chang
MS, National Taiwan University - 2006


Thesis Advisor: Vasily M. Studitsky, Ph.D.
Graduate Program in Cellular & Molecular Pharmacology

Rutgers RWJMS Research Tower
Pharmacology Department 4th floor Conference Room
Piscataway

Wednesday, April 23, 2014
2:00 p.m.


Abstract

Maintenance of nucleosomal structure in the cell nuclei is essential for cell viability, regulation of gene expression and normal aging. DNA transcription and DNA replication are the two key processes that remodel chromatin structure in the nuclei.
Nucleosomes form a strong barrier and remain associated with DNA during transcription by various RNA polymerases. Our previous data identified a key intermediate (a small intranucleosomal DNA loop, -loop) that is likely required for nucleosome survival at the original location during transcription by RNA polymerase II (Pol II) through chromatin, and suggested that strong nucleosomal pausing guarantees efficient nucleosome survival. My current DNase I footprinting, protein mutagenesis and molecular modeling studies suggest that efficient -loop formation and nucleosome survival are mediated by electrostatic interactions between the largest subunit of Pol II and core histones. The height of the nucleosomal barrier to transcription correlates with the net negative charges of the histone-interacting Pol II surfaces. During transcription by RNA polymerase III, less efficient formation of the -loop was observed, most likely due to steric interference between the RNA polymerase and DNA in the -loop, resulting in nucleosome translocation along DNA. Overall, my studies suggest that the structure of the critical intermediate formed during transcription through the +45 region of nucleosomal DNA dictates the primary outcomes of this process (the rate and efficiency of transcription through chromatin, and the nucleosome fate on transcription). The data provide molecular explanation for chromatin dynamics observed on Pol II- and Pol III-transcribed genes in vivo.
The mechanism of replication by T7 replisome through a single positioned nucleosome was determined using newly established highly purified experimental system that recapitulates nucleosome survival observed during DNA replication in vivo. Nucleosome forms a strong barrier for replication. Parental nucleosomes survive at the original position on DNA with ~50% efficiency, and are displaced from remaining DNA templates. The exonuclease activity on T7 DNAP increases the replication processivity and decreases the efficiency of nucleosome survival. The data suggest a new mechanism for epigenetic inheritance of histone modifications.


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