Damage to DNA is the driving force in cancer and aging in humans and is caused spontaneously by byproducts of cellular metabolism in addition to exposures to a wide range of environmental agents. Humans possess different systems of proteins that physically interact with DNA to remove damaged nucleotides and replace them in an ordered multi-step process called DNA repair. Of these repair systems, base excision repair (BER) plays a role in removal of the most frequent types of DNA damage. However, in the cell, the DNA is associated with the DNA-organizing proteins of chromatin, called histones that directly prevent access to damaged nucleobases by repair enzymes. Though there are known mechanisms used by the cell to modify histone-DNA interactions, including post-translational histone modifications, it is unknown which, if any, of these histone changes are associated with the essential process of BER. We hypothesize that the impediment to BER caused by histones is, at least partially, overcome by particular histone posttranslational modifications. We have established protocols for creating a nucleosome core particle (NCP) model, consisting of the canonical histone octamer with associated DNA containing damage (in the form or uracil residues), which has been used as a prototype lesion to assess BER activity with purified repair enzymes. We propose to develop this model into a novel system which can be employed to directly assess the nature and necessity of histone modifications associated with the process of BER in cell nuclear extracts. To determine histone modifications associated with the distinct steps of BER, we will use biotin-tagged DNA in uracil-containing NCPs to physically retrieve the histones associated with damaged DNA at distinct incubation times in cell extracts. Assessment of the modifications associated with the retrieved histones will provide a unique method for determining, for the first time, the changes to histones associated with the process of BER (Aim 1). We will also measure the repair in cell extracts of uracils in NCPs consisting of histones lacking the peripheral domains entailing most posttranslational modification sites, a.k.a. "tail-less" histones, determining, for the first time, the necessity of histone tail modifications for the process of BER (Aim 2A). In addition, NCPs will be assembled from histones with alanine substitutions at lysine residues known to be targeted for acetylation after cellular treatment to DNA-damaging agents, as well as at candidate residues found to be modified in Aim1, providing us with a method to systematically examine the necessity of specific damage-associated modifications in BER (Aim 2B). These experimental results will serve as a basis for a new direction in the study of histone modifications associated with any DNA substrate and any chemical modification. Understanding the nature of histone modifications in response to DNA damage and during the process of repair are essential for determining the fundamental biological processes of repair regulation by chromatin and paves the way for developing clinical methods for carcinogen exposure assessments, cancer prevention and treatment.
DNA damage is the driving source of mutations and cancer, and repair of these lesions is restricted by compaction of the DNA into chromatin. This study will determine the spectrum and necessity of modifications to histone packaging proteins associated with the fundamental process of base excision repair. Thus, the project results have implications for the broad spectrum of cancer etiology, prevention and treatment, and help provide a rational basis for regulatory policies associated with environmental exposure to DNA damaging agents.
|Hinz, John M (2014) Impact of abasic site orientation within nucleosomes on human APE1 endonuclease activity. Mutat Res 766-767:19-24|