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DNA Structure and Replication

Introduction : •DNA replicates, and contains information for protein synthesis . •The investigator Miescher first isolated DNA in 1869 .•Garrod in 1900 Linked heredity to enzymes . •Hershey and Chase in 1950 determined that part of virus that infects bacteria and replicates is its nucleic acid and not its protein (figure 1a) the virus transfers DNA and not protein to the bacterium , therefore DNA is the genetic material . •Chargaff et.al (1909 – 1950) discovered DNA components , proportions, and positions• Watson and Crick (1953) elucidated DNA three dimensional structure . DNA is a Double Helix .


Characteristics of the Genetic Material The molecule to serve as the genetic material it must possess four major characteristics : 1. Replication and transmitted from generation to generation . 2. Storage of information 3. Expression of information 4. Variation by mutation

Human Genome :

Human genome contained in every cell and consist of : 23 chromosome pairs and a small mitochondrial DNA . 2 meters of DNA 3 billion DNA subunits ( the bases A, T, C, G ) Approximately 30000 genes code for proteins that perform most life function . Genes only make up ( 5 % ) of human genomic DNA and the rest is junk DNA . Genes consist of exon ( protein coding portions ) and intron (non coding regions ) . ( 99.7 % ) of human genomes are identical . ( 0.3 % ) of human genomes are different .

Chromosome :

The structure by which hereditary information is transmitted from one generation to the next . Located in the nucleus, it is consists of tightly coiled thread of DNA with associated proteins and RNA . The genes are arranged in linear order along the DNA . Chromosome consist of two chromatids that held together by centromere.


Chromatin : DNA and its associated proteins .Euchromatin : Parts of chromosomes that do not stain and that contain active genes .Heterochromatin : Dark staining chromosome parts that have few protein–encoding genes .

Chemical Composition of Eukaryotic Chromosomes :

1- DNA 2- Proteins ( Histones and non Histone Proteins ) 3- RNA in small amountsHistones : Group of proteins rich in basic amino acids ,( positively charged ) proteins , thus facilitating their binding to the negatively charged DNA . They function in the coiling of DNA in chromosomes and in the regulation of gene activity .Non Histone Chromosomal Proteins : All of the proteins in the chromosomes except the histones . It fall into several functional categories : scaffold proteins and chromatin – bound enzymes and transcription factors.


Forms of Chromosomes :
Chromosomes can be classified according to the Position of Centromere into : ( Figure 5 ) 1- Metacentric : The Centromere in the Middle of Chromosome . 2- Submetacentric : The centromere between middle and end of chromosome 3- Acrocentric : The Centromere very near the end of Chromosome . 4- Telocentric : The Centromere at the end of Chromosome .

ANATOMY OF MITOTIC CHROMOSOMES FROM HIGHER EUKARYOTES

DNA Organization in Eukaryotic Chromosomes :
DNA of human cell is 2 meters in length while the nucleus is a 5-10 micrometers, therefore DNA wraps at several levels until it is compacted into chromosome to fit in the cells nucleus . First level : Nucleosomes ( Beads on a String ) DNA coils around proteins called histones forming a beads on a string structure , the bead part is called Nucleosome . Nucleosome : a unit of chromatin structure consists of 146 Nucleotides of DNA wrapped around Octamer of histone ( two molecules each of H2a , H2b , H3 , H4 ) . This structure produces 10 nm chromatin fiber .

Figure 6 : Levels of DNA organization in eukaryotic chromosomes

Second level : Solenoid or 30 nm chromatin fiber : 10 nm chromatin fiber folds into 30 nm chromatin fiber or solenoid ( each turn of solenoid includes six nucleosomes ) with aid of H1 . Third level : Radial loop domains : Model for the organization of 30 nm chromatin fiber into looped domains , which seems to compact the DNA to 300 nm chromatin .

Fourth level : Metaphase chromosome : 

Chromatin is looped and attached to a non histone protein scaffold and further compaction of all radial loops to form metaphase chromosome . Figure 7

DNA Structure :

DNA : Deoxyribonucleic acid : is composed of two chains of repeating nucleotides . Nucleotide is building block of DNA consists of : 1- Phosphate group 2- Five carbon sugar : Deoxyribosr sugar 3- Nitrogenous bases : Adenine (A) and Guanine (G) are purine which have a two ring structure Cytosine (C) and Thymine (T) are pyrimidine which have a single ring structure . Figure 8



Characters of DNA :
1- Double Helix : Watson and Crick ( 1953 ) proposed that DNA exist as a Double Helix in which the 2 Polynucleotide chains (strands) are coiled a bout one another in a Spiral . (Figure 9 ) . Each strand consist of a sequence of Nucleotides linked together by Phosphodiester bonds , joining adjacent Deoxyribose moieties . The 2 strands are held together by Hydrogen bonding between bases in opposing strands .

2- Complementary Base Pairing : The Rule of Complementarity is :

Adenin pairs with Thymine by 2 Hydrogen bonds , and Guanine pairs with Cytosine by 3 Hydrogen bonds . Once the sequence of bases in one strand of a DNA is known , the sequence of bases in the other strand is also known because of the specific base- pairing . this property complementarity of the 2 strands that makes DNA uniquely suited to store and transmit genetic information . Figure 10


3-Antiparallel : Antiparallelism in a DNA molecule arises from the orientation of the deoxyribose sugars . One strand of the double helix runs in 5 to 3 direction, and the other strand runs in 3 to 5 direction .

Gene : is acertain segment of DNA that contains the necessary code to make RNA or polypeptide (proteins). Figure 12 Gene accomplish their function through replication, transcription and translation
4- DNA carry negative charges because of their phosphate groups . 5- DNA is the same in all nucleated cells of persons body .

Gene

DNA – Replication : DNA replication occur during S phase of cell cycle . During Replication the parental strands unwind, and each parental strand provides template for DNA polymerase to bind complementary bases : A with T and G with C . The new result of Replication is thus 2 progeny DNA molecules identical to the parental Double Helix . Figure 13

Table 1 : Enzymes involved in DNA Replication and Their Function

Enzymes
Functions
Helicase
unwinds parental double helix
Binding proteins
stabilize separate strands
Primase
adds short RNA Primer to template strand
DNA polymerase
Binds nucleotides to form new strands Proofreading activity checks and replaces incorrect bases Removing RNA primer Because DNA Polymerase added Bases in 5 to 3 direction only , and DNA is antiparallel , one Strand is synthesized continuously in 5 to 3 direction and called Leading strand . wherease the opposite strand is synthesized discontinuously by short segments of DNA called Okazaki Fragments that are joined together by ligase and called Lagging strand .


Ligase
joins Okazaki fragments , and seals other nicks in sugar phosphate backbone . ( figure 14 & 15 )

Transcription and Translation

RNA Structure:
The main differences between DNA and RNA ( Ribonucleic acid ) illustrated in (table : 2) and figure : (16 )

Table 2 : The main differences between DNA and RNA

RNA
DNA
1-Usually single stranded 2-Uracil as a base ( figure 2 ) 3-Ribose as the sugar4- Functionally : carries protein encoding information and controls how information is used . 5- Transient : the presence of the –OH of the ribose makes RNA much less stable than DNA , which is critical in its function as a short-lived carrier of genetic information . 1-Usually double stranded 2-Thymine as a base 3- Deoxyribose as the sugar 4- functionally : stores RNA and protein encoding information, and transfers information to daughter cells 5- Persists

Types of RNA:

1-Messenger RNA ( mRNA ) : Encodes amino acid sequence . Each three mRNA bases in a row form a genetic code word or codon that specifies a certain amino acid . 2-Transfer ( tRNA ) : Transports specific amino acids to the ribosome for protein synthesis . 3-Ribosomal ( rRNA ) : Associates with proteins to form ribosomes which structurally support and catalyze protein synthesis . ( figure 17 )

Types of RNA

Transcription ( RNA synthesis ) :
Process through which RNA is formed along a DNA template . The enzyme RNA polymerase catalyzes the formation of RNA from nucleotides . Transcription occurs in three stages : 1- Initiation : The initiation of transcription by RNA polymerase requires the assistance of several basal transcription factors : A protein that regulates the transcription of genes . Figure 18


Initiation
The initiation of transcription by RNA polymerase requires the assistance of several basal transcription factors : A protein that regulates the transcription of genes .

Transcription ( RNA synthesis ) :

2- Elongation : RNA polymerase unwind the DNA double helix locally, RNA polymerase then adds RNA nucleotides to a growing chain, in a sequence complementary to the DNA template strand ( Uracil in place of Thymine) RNA is transcriped from the template strand of DNA . The other DNA strand is called the coding strand . ( figure 19 & 20 ) 3- Termination : A termination sequence in the gene signals the end of transcription .

mRNA Processing in Eukaryotes

Genetic Code:
The linear sequences of 3 Nucleotides in mRNA that Code for a sequence of amino acids in Polypeptide chain during the Process of Translation at the Ribosome . 64 Codons more than enough to Code for 20 different amino acids found in Proteins. (Figure 22).

Features of The Genetic Code :

1- Genetic Code is Triplet : AAA Code for Lysine . 2- Unambiguous : each Triplet Codon has only one meaning . 3- The Genetic Code has start and stop signals . There is one start signals ( AUG ) . and there is 3 stop signals ( UAA , UAG , UGA ) . 4- Universal : the Code is same and stable in all living organisms . 5- The genetic code is nonoverlapping and Degenerate .

Genetic Code :

Translation ( Protein Synthesis )
The Process during which the genetic information ( which is stored in the sequence of Nucleotides in an mRNA molecule ) is translated , according to the specification of the genetic code into the sequence of amino acids in the Polypeptide gene product . A ribosome has two subunits small subunit and large subunit . Eukaryotic ribosome ( 80 S ) has small subunit ( 40 S ) and large subunit ( 60 S ) .


Protein synthesis consist of 3 phases : 1- Initiation Phase : A small ribosomal subunit binds to mRNA : an initiator tRNA with the anticodon UAC pairs with the start codon AUG . ( Figure 23A ) . The large ribosomal subunit completes the ribosome , initiator tRNA carry methionine occupies the P-site the A-site is ready for the second tRNA The small and large subunit together form two tRNA binding sites P (Peptidyl ) site and A ( Aminoacyl ) site .

1- Initiation Phase :

2- Elongation Phase : Elongation consist of 3 steps : 1st- A second charged tRNA with an anticodon complementary to the second codon on mRNA binds to A-site . 2nd- Peptide bond formation : Peptidyl Transferase ( part of the large ribosomal subunit ) can catalyze formation of peptide bond between the amino acids carried by the two tRNAs . It also disconnects methionine from the initiation tRNA as a result the tRNA at the A-site now carries two amino acids . 3rd- Translocation : Three concerted movements occur , collectively called translocation : 1- Uncharged -tRNA leave the P-site . 2- The dipeptide-tRNA in the A-site moves to the P-site . 3- And the ribosome moves a long the mRNA by three nucleotides ( codon ) toplace the next codon in the A-site . The empty A-site now receives another tRNA whose identity is determined by the next codon in the mRNA and the ( peptide bond formation and translocation ) occurs once again . ( Figure 23B ) .

2- Elongation Phase :

3- Termination Phase : Termination of protein synthesis occurs when one of 3 stop codons ( UAG , UAA , UGA ) appears in A-site of the ribosome . A protein called release factor recognize stop codons and hydrolysis the bond between the last tRNA at the P-site and the polypeptide releasing them . The ribosomal subunits dissociate . ( Figure 23C ) . The resultant polypeptide chain may be enzyme , hormone , antibody , or structural proteins .

3- Termination Phase :

The Central Dogma of Molecular Genetics :
Is that Genetic information is Transferred from : 1- DNA to DNA through Replication . 2- DNA to RNA to Protein during Gene expression . ( Figure 9 ) . The Transfer of genetic information from DNA to RNA to Protein or Protein Synthesis involves : Transcription : The Transfer of genetic information from DNA to RNA . Translation : The Transfer of genetic information from RNA to Protein.