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4. Beskrive hvordan følgende elementer er involveret i DNA replikation: template, primer, substrater (deoxynucleosid triphosphater, dNTPs), primase, DNA polymerase I og III, DNA ligase, leading og lagging strands (Okazaki fragmenter) og diskontinuerlig syntese,
 Devlin, s.163-169, fig.4.2
Stryer, s.750
Stryer, s.761

 

Template - A sequence of DNA or RNA that directs the synthesis of the complementary sequence.  

Primer - short stretches of RNA (ca. 8-10 nucleotides long) that are already base-paired to the template and provide the free 3`-OH to which the nucleotides are added by DNA polymerases. The primers are synthesized by primase. They are found in between the Okazaki fragments of the lagging strand and on the beginning on both daughter strands.  

Primers are necessary because the DNA polymerase does not join the first two nucleotides to start a strand. The requirement for a primer is not due to the chemistry of phosphodiester bonds, since RNA polymerases can start a strand on their own.

The likely reason is low accuracy of proofreading preformed by DNA polymerase on the first few nucleotides in the sequence. The possibility to identify and remove the primer later, as well as fill the gap more accurately contributes to less errors in DNA synthesis.

 The primer can be removed by DNA polymerase because of its 5`-3`activity. This enzyme can also fill in the gap by adding nucleotides, while DNA ligase seals the nick between the newly added DNA sequence and the DNA that already was there.

Primase - an RNA-polymerase that synthesizes the primer sequence using ATP, UTP, GTP, CTP.

Substrates
Devlin, s.164 - fig.4.2

Substrates - the building blocks of DNA are deoxy - nucleoside triphospahts (5`-dNTP) that lie around in the nucleus of the cell: dATP, dTTP, dGTP, dCTP. They are the precursors of the DNA nucleotides, because the addition of a mononucleotide to a growing chain is not a spontaneous process.  

This entire DNA-synthesis process is catalyzed by DNA polymerases. When a deoxy-nucleoside triphosphate incomes, it first forms a hydrogen bond with the complementary base and only then the DNA polymerase links the incoming base with the predecessor in the chain by a nucleophilic substitution reaction.

The phosphodiester bond of the dNTP connecting the first (α) phosphate attached to 5`-carbon of the deoxyribose and the two other ( β,γ ) phosphates undergoes a nucleophillic attack from the free 3`-OH group of the previous nucleotide attached to the DNA. That way, a new bond is created between the a  phosphate of dNTP and the  3`-OH group of deoxyribose.

The terminal two phosphates are released as inorganic phosphate and hydrolyzed in the cells. This makes the reaction irreversible.

 DNA polymerase I:

 

  1. polymerase activity - fills the gaps left after fx. the RNA primer is removed. A gap means that at least one nucleotide is missing.

  2. 5´- 3´ exonuclease activity - it can remove the RNA primers of the lagging strand and act as a repair system

  3. 3´- 5´exonuclease activity - proof reading, removes the wrongly incorporated nucleotide at the 3`-OH end

  4. high processivity - lets go of the DNA molecule after adding 20 nucleotides

  5. low speed - adds 10 nucleotides/s

DNA polymerase III:
 Stryer, s.763-764, fig. 27, 31;27.33

  1. The replication unit of DNA polymerase III is a asymmetric dimer. It is structured as a dimer in order to be able to replicate both parental DNA strands in the same place at the same time. It is symmetric because the leading and the lagging strands are synthesized differently.

  2. Polymerase activity - synthesizes DNA by adding dNTP in the 5´-3´direction, meaning adds nucleotides to a free 3´- OH group. Is not used to fill in gaps.

  3. no 5´-3´ exonuclease activity - meaning it can not remove the RNA primer and it can not act as a repair system

  4. 3´-5´exonuclease activity - proof reading, removes the wrongly incorporated nucleotide at the 3`-OH end

  5. extremly high processivity - the processivity unit of DNA polymerase III is formed as a star-shaped ring. In the center of the ring there is a hole that can readily accommodate a duplex DNA molecule, yet leaving enough space between the DNA and the enzyme to allow rapid sliding and turning during replication. The DNA polymerase does not let go of the DNA until the entire replication is finished

  6. high speed - adds 1000 nucleotides/s

DNA ligase
Stryer, s.761

DNA ligase - after the RNA primer is removed and the gape is filled by DNA polymerase I, there is still a nick left which is sealed of by DNA ligase. A nick is an interruption in the phosphodiester backbone with no missing nucleotides.

DNA ligaze catalyses the formation of a phosphodiester bond between the 3`-OH group at the end of one DNA molecule and the 5´-phosphate group at the end of the other. DNA ligaze can not add nucleotides to a single stranded DNA, it needs a double helix in order to be operational.

The energy source in the eukaryotes is ATP, which is cleaved to AMP and PPi; in prokaryotes NAD+ which breaks to AMP and NMN.

Leading strand - the daughter strand of DNA that is replicated continuously from the origin of replication in the direction 5´-3´. It has its 3´-end towards the replication fork and the DNA polymerase III has not problems adding nucleotides on the free 3´-OH group - continuous replication.
 

Lagging strand - the strand which has its 5´-end towards the replication fork. The DNA polymerase adds nucleotides only in 5´-3´direction, while the lagging strand has the 3´-5´ direction, meaning has a free 5`-OH group.

That is why this strand is synthesized in small segments, called Okazaki fragments, after the template DNA for the lagging strand makes a loop, which allows the DNA polymerase to add nucleotides in the 5´-3´direction. The gaps left after the removal of the primers are filled by DNA polymerase I.

The synthesis of the lagging strand is therefore said to be discontinuous.

 

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