Date of Award

8-1-2012

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

BHAUMIK, SUKESH

Abstract

DNA is the storehouse of genetic information for the normal functioning of all cellular processes. This DNA is "read" by RNA Polymerases (RNAP) to carry out the transcription process, which is one of the most vital regulatory mechanisms within the cell for gene expression. There are three important RNA Polymerases available in the cell. RNAPI synthesizes ribosomal RNAs, while RNAPII makes mRNA precursors, snRNAs and most microRNAs, additionally RNAPIII takes care of tRNAs, small RNAs and 5S rRNAs that occur in the nucleus and the cytosol. The work in our lab focuses on the function of RNAPII transcription. It is not straightforward for RNAPII to transcribe the DNA, it needs to struggle with two major roadblocks that are in its path. First, is the nucleosomal barrier that is scattered across the coding regions of active genes. The mechanism to overcome this major obstacle is a very complex process that involves an extensive range of proteins which work collectively to help in the alteration of chromatin structure that ultimately facilitates transcriptional regulation. The second obstacle is that the DNA is also subject to constant onslaught by a variety of intrinsic and external damaging agents. Maintenance of normal physiological functions in addition to cell survival depends on the ability to preserve the genome integrity by protecting it from different mutations. Consequently, the cells have developed an extremely evolved repair mechanism known as the DNA damage response (DDR) system. This DDR pathway can restore various forms of damaged DNA that encompasses single base modifications, single strand damages and even the most toxic double strand breaks. Transcription-coupled DNA repair is involved in taking care of the single strand damages that can be induced by various chemicals including 4-nitroquinoline 1- oxide (4NQO) which forms bulky adducts on the DNA bases. On the other hand, double strand breaks are repaired via more complex and involved mechanisms that include non-homologous end joining and homologous recombination. Once the DNA is protected from injury it is prepared for transcription, replication and recombination. In my thesis, I have attempted to take a look at this intricate network and have studied the function of Rad26p, in yeast (associated with CSB-Cockayne syndrome B, in humans), a transcription coupled DNA repair factor, having an additional role in transcription. The results reveal that Rad26p plays a novel role in the regulation of elongation by modulating histone H2A-H2B dimer occupancy. Further, the study revealed how Rad26p is targeted to lesions for DNA repair. Next, I extended my study to the role of a nucleotide excision repair factor, Rad14p (associated with Xeroderma Pigmentosum, XPA- in humans), and for the first time demonstrated its function in transcription initiation in the absence of DNA damage. Finally, I analyzed whether repair of double strand breaks could be connected to transcription via an elegant method developed in my lab. This presented us with first time evidence that establishes that double strand break repair is faster on actively transcribing gene (ADH1) when compared to a nearly silent gene (MATα locus); thus, supporting our transcription-coupled DNA double strand break repair. The significance of all the proteins involved in my study is reflected by the various syndromes and diseases that arise due to mutations in their respective human homologues. Therefore, the results of my study will significantly advance our understanding of the regulatory mechanisms that are involved in the normal physiological functions of the cell which can be extended to humans.

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