PROCESS OF DNA SYNTHESIS PLUS ALL 3

3.1 Pick buffy coat aliquots for DNA extraction Buffy coat was picked from storage in preparation for DNA extraction.DNA replication steps. The following sections describe in detail the processes undertaken to prepare DNA for genotyping. UK Biobank was responsible for sample retrieval, DNA extraction and shipping of samples for genotyping.

Process Of Dna Synthesis Plus All 3

In E coli, replication origin is called OriC which consists of 245 base pair and contains DNA sequences that are highly conserved among bacterial replication origin. Termination DNA replication in prokaryotesDNA replication begins from origin. There are 2 processes in protein synthesis: Transcription (DNA makes all 3 forms of RNA in the nucleus) Translation (DNA plus all 3 forms of RNA together make proteins at the ribosome in the cytoplasm) Transcription (DNA makes all 3 forms of RNA in the nucleus)The process starts with a short strand of DNA that binds by pairing its nucleotide bases to those in the DNA strand to be replicated. It proceed from a specific point called originThe repairing is really the process of replication. Transcription When a gene is to be expressed, the base sequence of DNA is copied or transcribed into mRNA. This process of protein synthesis occurs in two stages - transcription and translation.

The dnaB-dnaC interaction causes dnaB ring to open which binds with each of the DNA strand. This binding is facilitated by another molecule called dnaC. Two copies of dnaB proteins (helicase) binds to 13 mer repeats. The dna A protein and ATP trigger the opening of 13 mer repeats froming open complex. About 20 molecules of Dna A proteins binds with 9 mer repeats along with ATP which causes DNA to wraps around dnaA protein forming initial complex.

Similarly, single stranded binding protein binds to th separated strand and prevents reannaeling of separated strand and stabilize the strand. This stress is relieved by the DNA topoisomerase (DNA gyrase) by negative supercoiling. The activity of helicase causes the topological stress to the unwinded strand forming supercoiled DNA. DnaB migrates along the single stranded DNA in 5’-3’ direction causing unwinding of the DNA. The binding of helicase is key step in replication initiation.

DNA polymerase III then adds the nucleotides at 3’end. Leading strand synthesis is more a straight forward process which begins with the synthesis of RNA primer by primase at replication origin. Primer is closely associated with dnaB helicase so that it is positioned to make RNA primer as ssDNA of lagging strand. In case of E.coli primer synthesized by primase starts with ppp-AG-nucleotide. So, at first primase synthesize 10☑ nucleotide (RNA in nature) along the 5’-3’ direction.

On this level the synthesis of each okazaki fragments seems straight forward but the reality is quite complex. At first RNA primer is synthesized by primase and as in leading strand DNA polymerase III binds to RNA primer and adds dNTPS. The lagging strand synthesized in short fragments called Okazaki fragments.

DnaG primase occasionally associated with dnaB helicase synthesizes short RNA primer. In elongation steps, helicasein front of primaseand pol III, unwind the DNA at the replication fork and travel along lagging strand template along 5’-3’ direction. DNA pol III use one set of core sub unit (Core polymerase) to synthesize leading strand and other set of core sub unit to synthesize lagging strand. Helicase (dnaB) and primase (dnaG) constitute a functional unit within replication complex called primosome. Both the strand are synthesized by a single DNA polymerase III dimer which accomplished the looping of template DNA of lagging strand synthesizing Okazaki fragments.

Both leading and lagging strand are synthesized co-ordinately and simultaneously by a complex protein moving in 5’-3’ direction. This initiates the synthesis of new Okazaki fragments. When the Okazaki fragments synthesis is completed, the replication halted and the core sub unit dissociates from their sliding clamps and associates with new clamp.

The gap is then sealed by DNA ligase using NAD as co-factor. The RNA primer are removed by exonuclease activity (5’-3’) of DNA pol-I and replaced by DNA. Lagging strand synthesis is not completes until the RNA primer has been removed and the gap between adjacent Okazaki fragments are sealed. Every so often the lagging strands must dissociates from the replicosome and reposition itself so that replication can continue.

Ter-TUS complex can arrest the replication fork from only one direction. Ter sequences function as binding site for TUS protein. The Ter sequence of 23 bp are arranged on the chromosome to create trap that the replication fork can enter but cannot leave. Coli chromosome meet at termination recognizing sequences (ter).

When either of the fork encounter Ter-TUS complex, replication halted. This seems the Ter-TUS sequences is not essential for termination but it may prevents over replication by one fork if other is delayed or halted by a damage or some obstacle. The other opposing replication fork halted when it collide with the first one.

Due to sheer size of chromosome in eukaryotes, chromosome chromosome contains multiple origin of replication. However pre-initiation occur in G1 pahse. The DNA finally transfer to two daughter cell.DNA replication in eukaryotes occur only in S-phase of cell cycle. Coli DNA topoisomerase IV (type II) cut the two strand of one circular DNA and segrate each of the circular DNA and finally join the strand.

It is known as licensing but licensed pre-RC cannot initiate replication at G1 phase. It occur in early G1 phase. 1 st stage requires, there is no CDK activities. The first steps is the formation of pre-initiation replication complex (pre-RC).

MEM complex is thought to be major eukaryotic helicase. After binding of ORC to origin, cdc6/cdc18 and cdtl coordinate the loading of MEM (mini chromosome maintainance) to origin. Furthermore ORC is analogue of prokaryotic dnaA protein. ORC is a hexamer of related protein and remains bounded even after DNA replication occurs. The replication begins with binding of ORC to the origin.

Exonuclease activity of DNA polymerase removes the RNA primer and polymerase activity adds dNTPs at 3’-OH end preceding the primer. At the end of DNA replication the RNA primer are replaced by DNA by 5’-3’exonuclease and polymerase activity of DNA polymerase ε. The leading and lagging strands are synthesized in the similar fashion as in prokaryotic DNA replication. DNA polymerase δ synthesizes and adds dNTPs at 3’ end of RNA primer. Again DdK and CdK recruit another protein called cdc45 which then recruit all the DNA replicating protein such that the origin get fired and replication begins. Then DdK phosphorylates MEM, which activates its helicase activity.

This gap must be filled before next round of replication. This missing DNA cause loss of information contain in that region. So, at 5’ end of each daughter strand there is a gap (missing DNA). When RNA primer at 5’ end of daughter strand is removed, there is not a preceding 3’-OH such that the DNA polymerase can use it to replace by DNA. But in eukaryotic organism with linear DNA, there is a problem.

These telomere sequence donot codes anything but it is essential to fill the gap in daughter strand and maintain the integrity of DNA.Telomere replication: end replication problem in Eukaryotic DNA Each species has its own species specific telomere repeats. Telomere in human cell consists of repeats of TTAGGG/AATCCC. These repeats of telomere sequence is different among different organisms. These sequence is known as telomere sequence.

First telomerase binds to 3’-OH end of parent strand by hybridization between its AACCCCAAC RNA sequences and TTGGGG DNA sequences (telomere sequences of T etrahymena). Unlike other DNA polymerase, telomerase adds DNA at 3’-OH end of parent strand not at the daughter strand and also it synthesizes the same sequences over and over in absence of template strand. Like other DNA polymerase, terlomerase also adds deoxyribonucleotide at 3’-OH end. Telomerase is a DNA polymerase (RNA dependent DNA polymerase) which adds many copies of telomere sequence at 3’-OH end of template strand.