Transcription, Translation, and Replication

DNA replication

the process by which a cell duplicates its DNA, replication ensures that each cell receives an exact copy of the genetic information. 

3 steps of Replication

1. Initiation: When replication occurs, happens at specific origins where DNA unwinds 

2. Primer synthesis: Primase synthesizes RNA primers on the template strand

3. Extenstion: DNA polymerase extends the DNA from the primer and replaces RNA primers with DNA later

DNA replication is semiconservative

each of the two identical daughter DNA molecules contains one parental strand and one newly synthesized strand; predicted by Watson and Crick, confirmed by Meselson and Stahl 

DNA is read in what direction and synthesized in what direction

DNA is read 3’->5’ and synthesized 5’->3’; The product of the new strand is opposite from that of the template strand

Replication Fork

DNA replication occurs here; where the double helix unwinds, and the new strands are synthesized ; unwinding of the parental strand and synthesis of new DNA occur simultaneously

Enzymes involved in DNA Replication

The primary enzyme involved is DNA polymerase, DNA polymerase adds nucleotides to the growing DNA strand. Other proteinssuch as helicase, primase, and ligase assist in unwinding DNA, initiating synthesis, and joining together the DNA fragments.

DNA polymerase 

Catalyzes nucleophilic attack by the 3′ hydroxyl at the primer terminus upon the α-phosphate of an incoming dNTP, base-paired with its templat e.The DNA polymerase synthesizes a new DNA strand/adds deoxyribonucleotides to a preexisting chain. DNA polymerase also have proofreading abilities that correct mismatches to ensure high fidelity in DNA replication

DNA polymerase reaction steps

1. DNA polymerase aligns an incoming deoxyribosnucloside triphosphase (dNTP) with the complementary base on the template strand. Ex. if wanting to pair with C dGTP will be used 

2. A phospodiester bond is formed between the 3’ hydroxyl group of the primer and the a-phosphate of the incoming dNTP. The step drives the release/hydrolysis of the phosphate (PPi) providing energy for this reaction 

   1. PPi+H2O-> 2Pi 

   2. Two energy-rich phosphates are expended per nucleotide incorporated.

3. The process continues with the addition of subsequent nucleotides, extending the DNA strand.

The overall DNA polymerase reaction is endergonic or exergonic 

endergonic (requires energy input) to synthesize the DNA strand, but it is coupled to the exergonic (energy-releasing) hydrolysis of nucleoside triphosphates (dNTPs). The energy from the breaking of the high-energy phosphate bonds in the dNTPs is used to fuel the endergonic process of forming the phosphodiester bonds between nucleotides

Polymerase chain reaction (PCR)

Used to amplify specific DNA sequences by using temperature cycling and DNA polymerase to make several copies for DNA recognition 

Polymerase used for PCR taken

From thermophilic bacteria (thermophiles) 

Steps of PCR: 

1. Denaturing: (98C), DNA is heated briely to separating DNA strands  

2. Annealing:  (55C), cooling allows primers to form hydrogen bond with ends of target sequence 

3. Extension: (72C) DNA polymerase adds nucleotides to the 3’ end of each prime

Sanger method

Method of determining DNA seqiecing; each ddNTP is modified with a fluorescent dye, each dye a different color; each fragment color based on the ddNTP in the sequencing and placed on one gel for analysis. Strand that shows up on analysis will be complementary DNA strand, had to be reversed to find analyzed strand.

Automated DNA sequencing

All four base-speficic termination reactions are analysed by use of ddNTPs and modified with dlurescent dyes; each ddNTP receives a unique color 

Origin of replication

Replication starts at this specific site, it occurs in both directions and forms replication forks that move away from the origin of the molecule. In eukaryotic cells DNA replication involves multiple origins of replication along the chromosomes. This allows a large genome to be replicated in a timely manner. 

Bidirectional Replication

Occurs in both prokaryotic cells and eukaryotic cells. Replication begins at a fixed origin, replication moves in opposite directions around the circle until they meet on opposite sides. 

High Fidelity of replication

DNA replication process is highly accurate and has proofreading mechanisms that ensure minimal errors.  

DNA replication full steps: 

1. Initation Replication begins at specific sites, origins. Proteins bind to region causing DNA to unwind and form a single stranded loop; this process is facilitated by helicases and other proteins that help DNA bending and unwinding

2. Primer synthesis: Priamse synthesizes short RNA primers on the single-stranded DNA. These primers provide a starting point for DNA polymerase to begin synthesis 

3. Elongagtion: DNA polymerase III, extends the DNA from the RNA primers. The leading strand is synthesized continuously, while the lagging strand is synthesized in short segments called okazaki fragments 

4. Primer removal and replacement: DNA polymerase I removes RNA primers and fills in the gaps with DNA nucleotides

5. Ligation: DNA ligase seals the nicks between Okazaki fragments, this completes the synthesis of the lagging strand 

6. Termination: Topoisomerase help in resolving the sinal structures, ensuring the replication process is complete.

Initiation in replication

Replication begins at specific sites, origins. Proteins bind to region causing DNA to unwind and form a single stranded loop; this process is facilitated by helicases and other proteins that help DNA bending and unwinding

Primer synthesis in DNA replication

Priamse synthesizes short RNA primers on the single-stranded DNA. These primers provide a starting point for DNA polymerase to begin synthesis 

Elongagtion in DNA synthesis

DNA polymerase III, extends the DNA from the RNA primers. The leading strand is synthesized continuously, while the lagging strand is synthesized in short segments called okazaki fragments 

Primer removal and replacement:

DNA polymerase I removes RNA primers and fills in the gaps with DNA nucleotides

Ligation in DNA replication 

DNA ligase seals the nicks between Okazaki fragments, this completes the synthesis of the lagging strand

Termination in DNA synthesis 

Topoisomerase help in resolving the sinal structures, ensuring the replication process is complete.

Helicase

unwinds DNA double strands

Primase

synthesizes RNA primers

Single stranded binding proteins 

prevent DNA duplex annealing

DNA Pol I

synthesizes DNA and removes RNA primers

DNA Pol III

the main force that synthesizes DNA

DNA ligase

links the Okazaki fragments

Topoisomerase

releases the tension of DNA duplex