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Rapid Histone Catalyzed DNA Lesion Excision and Accompanying Protein Modification in Nucleosomes and Nucleosome Core ParticlesDiksha JainKarpagam Sudhakar

IntroductionThe initial step of BER involves enzymatic activities of DNA glycosylases that process the N-glycosylic bonds linking the target bases and their deoxyribose sugars The central intermediate is an abasic [apurinic apyrimidinic (AP)] site in DNAIn a given day, each cell produces between 10,000 and 50,000 AP lesionsOxidized abasic sites eg. C4-AP, L, DOB arise following hydrogen atom abstraction from the 2'-deoxyribose ring of a nucleotide

Base excision repair is the most important cellular defense mechanism that evolved to avert the deleterious effects of the damaged bases in DNA. It has been estimated that AP site formation through the spontaneous hydrolytic loss of purines generates some 10 000 AP sites per day in a mammalian cell. Rest is the work of DNA glycolsylases. 2

2-deoxyribonolactone (dL) which is generated by initial hydrogen abstraction from the deoxyribose C1 carbon, followed by O2 addition and base loss3

C4-oxidized abasic site (C4-AP), 2-Deoxyribonolactone (L), 5-(1,4-dioxobutane) (DOB)4

Produced by -radiolysis , cytotoxic antitumor agents, such as bleomycin and the enediynes (e.g. neocarzinostatin)AP, C4- AP, and DOB yield DNA interstrand cross-links, A number of cytotoxic pharmacological agents, such as nitrogen mustards and mitomycin CC4-AP, DOB, and L inactivate base excision repair enzymesHistone proteins also catalyze excision of the electrophilic lesions, AP, C4-AP, DOB and L

Some of these cross-links are misrepaired by bacterial nucleotide excision repair in vitro and converted into double strand breaks.What I am trying to explain here is, these AP sites are naturally formed as an intermediate in the repair pathway, and also induced by various agents. Now, if they get repaired, they are not an issue. But when they are not repaired, they are mutagenic.5

Activated FeBLM abstracts a H atom from the C-4position of deoxyribose at the primary site (typically 5-G-Py-B-3 of one strand of the hairpin DNA, where B = any nucleobase), producing an AP (apyrimidinic/apurinic) site (I)6

AP forms ICLs selectively with the dG in 5'-C-AP , sequences under mild reducing conditions (NaBH3CN) 7

the presence of a 1,4-dicarbonyl functional group in DOB, which reacts rapidly with primary amines dramatically alter the outcomes of interactions. Covalent modification of Lys84 (9) and indirect evidence for reaction at Lys72. Both lysines have been implicated in Schiff base formation during 5'-dRP excision8

Chromatin is composed of monomeric nucleosome units. In each nucleosome DNA (~145 bp) wraps around an octameric core of 4 different highly positively charged histone proteins H2A, H2B, H3 and H4The histones also contain lysine-rich termini (tails) that protrude through the nucleosome core particle (NCP) Post-translational modification of the lysines in the conformationally mobile tails plays an important role in gene regulationHistone tails have been shown to catalyze the excision of AP sites almost 500 fold compared to free DNA

Now, the difference between a NCP and Nucleosome is that nucleosome has 1 linker DNA attached9

These tails are subjected to various modifications such as acetylation, methylation, phosphorylation, ubiquitination etc. This either creates sites for the recruitment of specific factors or modify existing sites so as to abolish previous interactions and thus alter the expression states.Acetylation (ac) occurs exclusively on lysine residues of histone peptides by Histone acetyltrasferases (HAT). Acetylation causes neutralisation of the positively charged lysine residues, hampering the histone-DNA interaction. Histone acetylation is predominantly associated with transcriptional activation.

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Crystal structure of the nucleosome core particle at 2.8 resolution.12

DOBMinor product of DNA oxidation resulting from H atom abstraction from C5` of DNA sugar backboneFormed at the 5` terminus concomitantly with a strand breakUndergoes -eliminationForms Interstrand crosslinks (ICLs)Also, irreversibly inhibits DNA Pol and

5-(2-phosphoryl-1,4-dioxobutane) (DOB)

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We are going to investigate the reactivity of DOB at specific sites within NCPs and Nucleosomes

Design and Preparation of NCPs and Nucleosomes containing DOBWidom 601 strong positioning sequences were used to bind to histone core because of strong affinityThey have the most flexible dinucleotide type, TA at minor groove-inward positions where DNA distortion is energetically most challenging and G|C-rich elements, which are predisposed to major groove compression, at major groove-inward positionsIndependent lesions were to be generated at specific sites SHL 1.5 (DOB89), SHL 4.5 (DOB119), and at SHL 0 (DOB 73)

Alignment of the highest affinity sequences derived by SELEX allowed Widom and colleagues to predict a consensus sequence for maximum affinity histone octamer binding over the central 73 bp of the nucleosome, this approach apparently did not allow full optimization of the outer H2AH2B binding sites. Oligonucleotides were synthesized on an Applied Biosystems Incorporated 394 oligonucleotide synthesizer15

Structure of one half of the NCP, as we can see there is only one round on DNA around the core. The position of each base pair with respect to the histoneoctameric core can be referred by its superhelical location (SHL), it is a translational parameter, relative to the dyad position (SHL 0), where the twofold axis of the octamer is. Each turn of the duplex corresponds to one unit change in the SHL. Herein, we describe the histone-catalyzed excision of DOB in nucleosome core particles (NCPs) and nucleosomes. Modified nucleotides are shown as spheres

The DNA at SHL 1.5 is bent, is a preferred binding site for DNA-damaging reagents and is close to the lysine-rich N-terminal tail of histone H4. DNAkinking in the region around SHL 4.5 severely disrupts base stacking.4 SHL 4.5 is also in close proximity to the N-terminal tails from histone H2A and H2B. The DNA is held more tightly at the dyad region (SHL 0), but is not as closely positioned to any N-terminal histone tails as at SHL 1.5 and 4.5.Modified nucleotides are shown as spheres16

Oligonucleotides containing DOB precursor at specific sites were generatedComplementary strands were also createdHybridization of the two strands, gave the ternary complex Internal radiolabeling and restriction enzymes site were incorporated at