Question

1. Develop a theory of bioenergtics constraints that drive the evolution of chromatin organization and mmechanisms of DNA replication. You must describe what the selective pressures that cause this adaptation and incorporate synthesis of general knowledge concerning DNA organization/function.

2. Compare and contrast the primary, secondary and tertiary structure of DNA and RNA in a cell.

3. Describe the multiple mechanisms of DNA damage surveillance and repair. If these exist, how do mutations still arise?

Answer 1

1) DNA replication must adapt to changes in chromatin organization associated with cell differentiation and development, whose deregulation can challenge genome stability and leads to mutations, cancer and many other genetic diseases. However, despite intensive studies, the mechanisms that coordinate where and when replication initiates in the human genome remain poorly known...control over the initiation of DNA replication, as well as the ability of the replication machinery to proceed during elongation through the multiple levels of chromatin condensation that are likely to be encountered, is known to involve the creation of chromatin accessibility. In the latter case, chromatin access will likely need to be a transient event so as to prevent total genomic unraveling of the chromatin that would be deleterious to cells. While there are many molecular and biochemical approaches in use to study histone changes and their relationship to transcription and chromatin accessibility, few techniques exist that allow a molecular dissection of the events underlying DNA replication control as it pertains to chromatin changes and accessibility.

Researchers addresses the ability of specific proteins to induce large-scale chromatin unfolding (decondensation) in vivo upon site-specific targeting to an engineered locus. Our laboratory has used this powerful system in novel ways to directly address the ability of DNA replication proteins to create chromatin accessibility, and have incorporated modifications to the basic approach that allow for a molecular genetic analysis of the mechanisms and associated factors involved in causing chromatin decondensation by a protein of interest. Alternative approaches involving co-expression of other proteins (competitors or stimulators), concurrent drug treatments, and analysis of co-localizing histone modifications are also addressed, all of which are illustrative of the utility of this experimental system for extending basic findings to physiologically relevant mechanisms. Although used by our group to analyze mechanisms underlying DNA replication associated chromatin accessibility, this unique and powerful experimental system has the propensity to be a valuable tool for understanding chromatin remodeling mechanisms orchestrated by other cellular processes such as DNA repair, recombination, mitotic chromosome condensation, or other chromosome dynamics involving chromatin alterations and accessibility.

2) DNA and RNA are different from their structure, functions, and stabilities. DNA has four nitrogen bases adenine, thymine, cytosine, and guanine and for RNA instead of thymine, it has uracil. Also, DNA is double-stranded and RNA is single-stranded which is why RNA can leave the nucleus and DNA can't. Another thing is that DNA is missing an oxygen.

RNA STRUCTURE

The primary structure denotes the ribonucleotide sequence (commonly referred to as base- sequence) of the mole cule. Usually, the base-sequence of an RNA molecule only consists of a combination of the bases A, G, C, U. Thymine and hypoxanthine are analogous to uracil (or adenine, respectively) and thus represented by their corresponding standard base. Furthermore, modified bases such as pseudouracil (Ψ) are represented by their most-similar standard base.

The secondary structure is formed by a subset of the cis- Watson-Crick/Watson-Crick base-pairs contained in an R NA molecule. This includes the standard (canonical) A-U and G-C pairings already known from the formation of DNA helices as well as the so-called G-U wobble-pairs. Successive base-pairs form energetically favourable and thus stable stem- regions. The unpaired regions between two stems are called loops. The secondary structure of an RNA molecule is formed formed by a number of secondary structure segments (motifs).

Base-pairs that do not belong to the secondary structure together with pseudo-base-pairs form the tertiary structure of the molecule. This includes other atomic interactions such as van der Waals forces, electrostatic and hydrophobic interactions and hydrogen-bonds between e.g. base and ribose residues. Tertiary contacts are interactions between distinct secondary structure elements. They induce local and/or global structure folds and as such are dominantly responsible for the overall three-dimensional structure of an RNA molecule.

DNA STRUCTURE

Primary structure of DNA is formed by the covalent backbone consisting of deoxyribo nucleotides linked to each other by phosphodiester bonds.  DNAs are long chains of nucleotide units or polydeoxyribonucleotides.  The substrates for polymerization are nucleoside triphosphates, but the repeating unit or monomer, of a nucleic acid is a monophosphate.  During polymerization, the 3’-OH group of the terminal nucleotide residue in the existing chain makes a nucleophilic attack upon the alpha phosphate of the incoming nucleoside triphosphate to form 5’à3’ phosphodiesterbond.  This reaction is catalyzed by DNA  polymerase.  Serial polymerization generates long polymers variously called chains or strands, containing an invariant sugar-phosphate backbone with 5’à3’ polarity and projecting  nitrogenous base.

Secondary = DNA molecule present as an alpha helical structure and if it is double stranded structure.  Here, the relatively hydrophobic bases are present inside the molecule which polar sugar and phosphoric acid molecules are present on the outside.  This shields hydrophobic bases from water and made hydrophilic sugar and phosphoric acid molecule to interact with water.

B) Two polynucleotide chains are associated by hydrogen bonds between bases.  The double helical structures is stablised by hydrophobic interactions between adjacent bases brought about by electrons in pi rings.  There are two hydrogen bonds between adenine and thymine.  Three hydrogen bonds present between guanosine and cyotosine.

Tertiary= Nucleic acid tertiary structures reflect interactions which contribute to overall three dimensional shape.  This includes interactions between different secondary structure elements, interactions between single strands and secondary structures and topological properties of nucleic acids.  Examples for tertiary include cruciform, triple helices and super coils.

3) Cells are equipped with intricate and sophisticated systems—DNA repair, damage tolerance, cell cycle checkpoints and cell death pathways—that collectively function to reduce the deleterious consequences of DNA damage.Cells respond to DNA damage by instigating robust DNA damage response (DDR) pathways, which allow sufficient time for specified DNA repair pathways to physically remove the damage in a substrate-dependent manner. At least five major DNA repair pathways—base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR) and non-homologous end joining (NHEJ)—are active throughout different stages of the cell cycle, allowing the cells to repair the DNA damage. A few specific lesions can also be removed by direct chemical reversal and interstrand crosslink (ICL) repair. These repair processes are key to maintaining genetic stability in cells.

but with the possibility of a concurrent introduction of an incorrect base that can be fixed into a mutation in the subsequent round of replication. Under the circumstances, when the damaged DNA persists, programmed cell death or apoptosis, a regulatory response to DNA damage, is activated to get rid of cells with extensive genome instability.

incorrectly paired/incorporated nucleotides that escape proofreading and MMR become mutations in the next round of replication and are a major source of spontaneous mutagenesis...In the case of deamination of cytosine, for instance, the native C:G base pairing alters to a U:A base pair in the first round of replication, which in the next round of replication results in a CG→TA mutation. Cytosine and 5-methyl cytosine are the most frequently deaminated, but 5-methyl cytosine is deaminated three to four times more frequently than cytosine...Consequently, the GC→AT transition at the CpG sequences accounts for one-third of the single site mutations responsible for hereditary diseases in humans