Replication in Prokaryotes and Eukaryotes

Replication

• One of the single strands of the double-stranded DNA molecules is the template for the generation of the other.

• DNA replication is semi-conservative. Each of the two molecules double-stranded molecules resulting from replication contains one old and one new single strand.

Proving How the DNA is Replicated

• Watson and Crick proposed the hypothesis of semi-conservative replication in their original Naturepaper.
• The Meselson Stahl experiment was a real classic in genetic research.
• It was a critical experiment by design. This means that no matter what the result, they would have their answer.

Meselson and Stahl Experiment

• E. coli are grown in heavy nitrogen (¹⁵N) for many generations.
• This caused the nitrogen in the DNA molecule of each cell to contain ¹⁵N, a heavier than typical isotope.
• The E. coli were then grown for one or two cell divisions in ¹⁴N, the lighter and typical isotope.
• DNA was spun in a cesium chloride gradient. Meselson and Stahl actually invented this technique, called density centrifugation, which now has many other applications, just for the purposes of this experiment.
• The cesium chloride gradient and centrifugation separates molecules based on their density.
  • The DNA molecules with ¹⁵N are more dense than those with ¹⁴N, and band below DNA with¹⁴N.
  • If two bands were observed after one division in ¹⁴N, there would have been wholly old strands and wholly new strands. This would have been consistent with and meant the replication was conservative.
  • If there was just one band after one division, replication could be either dispersive or semiconservative.
• The result was just one band after one division.
  • If one or a long smear was observed after two divisions in ¹⁴N containing medium, dispersive replication would have been the mode.
  • If intermediate weight and light weight molecules were found, semiconcervative would be the mode.
• This is what was found; the replication was semiconservative. This was the predicted outcome of Watson and Crick.

DNA Replication: A Closer Look

• DNA polymerase enzymes are involved in repair and chromosome replication.
  • Prokaryotic DNA I: Discovered by the famous Dr. Authur Kornberg - this enzyme polymerizes small DNA segments during replication and repair. Most of the DNA polymerase found in prokaryotic cells is DNA polymerase I.
  • DNA III: This is the primary DNA polymerase involved in DNA replication of bacterial chromosomes.
• DNA always replicates from 5' to 3' in direction using the anti parallel strand as a template.
  • Therefore, for each double-strand of DNA, one strand must replicate in the opposite direction of the other.
  • Why only in the 5' to 3' direction? Because a new base can only be added to the 3' OH position.
  
• Also, a new base can only be added to an existing polymer!
  • So how does it start? (This question is answered later in this unit.)
  • A special DNA template dependent RNA polymerase called primase makes a stretch of RNA that complements the template strand and forms a RNA / DNA duplex.
  • Next, DNA polymerase adds to that RNA molecule.
  • Later, the RNA molecule is removed.

DNA Replication Steps

• Initiation: this involves the assembly of a replication fork (bubble) at an origin of replication sequence of DNA found at a specific site of the circular chromosome of a bacterium. The fork is generated by a complex of proteins called a primosome.
• Elongation: this is the addition of bases by another complex of proteins called the replisome. Parental strands unwind and daughter strands are synthesized.
• Termination: the duplicated chromosomes separate from each other. This is actually a complicated procedure, the details of which we won't cover in this course.

       

DNA polymerase

• Some types of DNA polymerase are occasionally called DNA replicases.
• Other types are considered DNA repair enzymes.
• All DNA polymerases polymerize a polynucleotide by adding to an existing double-stranded stretch of DNA.
• Aggregates of specific subunits of polypeptides makeup the replicase molecule.
• In E. coli, there are three major DNA polymerases: DNA polymerase I, II and III.
• DNA poly I is found in the highest concentration of all DNA polymerases; it is involved in DNA repair and assists with primary DNA replication.

• DNA poly II is exclusively involved in repair.

        

• DNA poly III is the major DNA polymerase role.

• All DNA polymerases add to the 3’ OH of the existing polynuceotide.
• DNA polymerases have other enzymatic activities as well.
  • One is an exonuclease activity, the digestion of the polymer into monomers, which is the reverse activity of polymerization.
  • One can diagram it to look a bit like PackMan of the video game. If the enzyme has exonuclease activity, it works in the 3' to 5' direction, which is the opposite of the 5' to 3' polymerase activity. Some DNA polymerase holoenzyme complexes have both a 3' to 5' and a 5' to 3' exonuclease activity. DNA polymerase I does for example.
  • This function is involved with error reduction: "proof reading". It is also involved with removal of the RNA primers and replacement of the RNA with DNA. There is also a lot of involvement of these activities in non-replicative DNA repair.
  • Another enzymatic activity associated with DNA polymerase is proofreading. This is a check that the correct base was incorporated immediately after the fact.

Fidelity of Replication

• The accuracy or fidelity of DNA replication can be measured relatively easily and has been for many different types of organisms.
• It can be expressed in a number of different ways, for example, errors per nucleotide incorporated.
• It varies substantially between prokaryotes, eukaryotes, unicellular and multicellular organisms.
• A typical value given by some genetics textbooks is 1 error/genome/1000 bacterial replications.
• It would be 10^-3 instead of 10^-8 to 10^-10, if there were no checks on the system.
• Checks include a pre-synthesis check to verify the incoming base is correct.
• The proofreading check verifies that the correct base was incorporated.
• The 3’ to 5’ exonuclease activity assists when it is found that the base incorporated was incorrect. It removes the incorrect base so that the polymerase can try again to get it right.
• The DNA polymerase I enzyme is 103 kD.
• Of this a 68 kD portion is called the Klenow fragment.
• Klenow has the 3’ to 5’ exonuclease and the polymerase activity in different regions of the fragment.
• Another 35 kD fragment has a 5’ to 3’ exonuclease activity.
• The E. coli DNA polymerase I incorporates about 10 bases in length at a time. This is called its processivity. Some organisms poly I incorporate more, some less.

Nick Translation

• Nick translation is an extremely useful technique for labeling DNA.
• First DNA is nicked using a shearing force such as forcing the solution containing the DNA molecules through a fine bore hypodermic needle.
• The DNA with nicks, DNA polymerase I and labeled deoxiribonucleoside triphosphates are combined in solution, and the DNA polymerase I molecules attach to the nick, use their 5’ to 3’ exonuclease activity to digest out existing nucleotides, while replacing them with labeled ones using their polymerase activity.
• This technique can be used to incorporate radioactive labeled nucs.

DNA poly III

• DNA polymerase III is the main polymerase for E. coli chromosome replication.
• It is made up of many subunits.
• It has several different activities.
• It is found in different forms ( subunit associations).
• E. coli's has 3’ to 5’ exonuclease proofreading.

  

• The gene dna E codes for the 130 kD fragment which has the polymerase activity.
• The alpha subunit works to increase error proofreading ability.

Types of Mistakes Made

• Frameshift(s) are extra or missing base(s).
  • Frameshifts are influenced by the processivity of the enzyme.
  • Slippage is less if processivity is greater (adding more bases before disassociating).
  • Strings of some bases, especially short repeats, can increase the frequency of frameshift mistakes.
• Substitutions are when the wrong nucleotide is incorporated.
• Substitution rates are strongly influenced by the proofreading function of the DNA polymerase.

Eukaryotic DNA Polymerase

• DNA Polymerase alpha and delta replicate the DNA.
• DNA polymerase alpha is associated with initiation, and delta extends the nascent strands.

      

• DNA polymerase epsilon and beta are used for repair.
• DNA polymerase gamma is for replication of mitochrondrial DNA.

Priming

• All DNA polymerases require a 3’ end of an existing double-stranded molecule to add nucleotides to.
• There are several different ways this is done.
  • Cells make RNA primers.
  • Some viruses use a preformed RNA.
  • A nick or gap in the phosphodiester backbone of the DNA molecule is used as the starting point.

Semidiscontinuous synthesis

• The replication fork moves in one direction.
• However, DNA is replicated in the 5’ to 3’ direction only.
• How can this be?
• There is a leading and lagging strand.
• Short pieces of DNA called Okazaki fragments with a short stretch of RNA at each 5' end are found temporarily on the lagging strand. 
• (The leading strand might at times appear to be fragmented also, but this is due to deoxi-uracil getting mistakenly incorporated instead of thymine, and then the uracil getting cut out by uracil glycosidase and AP endonuclease.)
• This system of replication is called semi-discontinuous because one strand, the leading, is continuous, and the other, the lagging, is discontinuous.
• Each Okazaki fragment starts with about 10 bases of RNA primer.
• What happens to Okazaki fragments? Does chromosomal DNA really contain RNA also?
• Ligase fails to seal gaps if the gap is between an RNA and a DNA base, which is how the gap exists between each separate Okazaki fragment.
• Time and the gap give DNA polymerase the opportunity to do the Pack Man routine.
  • DNA polymerase I eats the RNA bases (exonuclease activity), whilst incorporating new DNA bases (DNA polymerase activity).
  • Actually, a DNA polymerase I molecule will digest and add a few bases, and then fall off.
  • Because a gap still exists, another DNA polymerase I molecule will do the same again.
  • If the gap is now between two deoxiribonucleotides, either DNA polymerase I or ligase will associate with the gap.
  • If ligase gets their first, it seals the gap.
  • Ligase will "get their first" sooner or later, depending on concentrations, and the gap will get sealed.
• DNA polymerase I is using the end of the last Okazaki fragment, which is composed of DNA, as its primer.
• DNA polymerase I removes and adds bases beyond the RNA primer stretch a bit, unnecessarily.
• Ligase seals the gap.
• Ligases use energy from NADH (E. coli) or ATP (T4 phage).

Primosome

• First, helicase separates the strands of the double-stranded DNA molecule; E. coli's DnaB codes for helicase.
• Single-strand DNA binding proteins maintain the strands in this region as single-stranded.
• This occurs at the oriC site, the bacterial origin of replication.
• Primase, the DNA template dependent, RNA polymerase, which makes the primers, is coded for by the dna G gene.
• Unwinding the DNA by helicase requires ATP as an energy source and substrate.
• A note on unwinding enzymes:
  • Topoisomerases effect the coiling of double-stranded DNA.
  • Type 1 work by making single-strand breaks; Type 2 work by making double-strand breaks.

Stopping

• The replication fork moves in opposite directions away from the origin of replication, forming two bubbles. 
• How does it know when to stop in bacteria, so that just one round of replication takes place, and you don't just get a jumble of many copies?
• In E. coli, there are ter elements, which are 23 base pair consensus sequences.
• These are binding sites for the tus gene protein.
• This stops the Dna B product, helicase, from unwinding DNA in the ter element regions.
• Strands synthesize just up to ter.
• The strand extension is prevented in one direction but can read through in the other direction until the bubbles meet. The two strands must then disconnect from each other.

Summary

• Only one strand can replicate continuously. The other is discontinuous.
• On the discontinuous strand, short segments are called Okazaki Fragments, these are about 1,500 bases in length in prokaryotes, and 150 bases in eukaryotes.
• The continuous is called the leading strand, the discontinuous is the lagging strand.
• The leading has high processivity, it continues until the entire strand is replicated.
• The lagging requires these repeated steps:
  • 1. Primer synthesis
  • 2. Elongation
  • 3. Primer removal
  • 4. Ligation (joining the pieces). 
• Where as DNA Poly III is involved in the leading strand synthesis and adding bases to the primers of the lagging strand, DNA Poly I later recognizes the RNA primers and removes them with its exonuclease activity and replaces the RNA with DNA.
• Subsequently, patches must be made. Because connection of the separately synthesized fragments do not involve incorporation of a nucleoside triphosphate, a special enzyme does this,Lygase.to put it all together.

Origins of Replication and Replication in Eukaryotes

• The DNA sequences required as origins of replication in eukaryotic cells are called Autonomously Replicating Sequences (ARS).
• They contain an eleven base pair core consensus sequence.
• Progress has been made toward the replication of the DNA of eukaryotics in test tubes using eukaryotic enzymes.
• Two different DNA polymerases are required.
• One for the leading and another for the lagging strand.
  • In prokaryotic cells rates of replication are 500 bases per second.
  • In eukaryotic cells rates are 50 bases per second. Eukaryotes have 100 to 3,000 times more DNA than prokaryotes.
  • Bacteria have only one ARS, but eukaryotes have many.
  • In eukaryotes, clusters of origins in certain regions of the chromosome are active at a given time.
  • Each origin is separated by 30,000 to 300,000 base pairs.
  • There is a repeatable order to the activation of each region of the chromosome.
  • Early to replicate regions include areas of euchromatin.
  • Late to replicate regions are heterochromatin.
  • There is a yet unknown mechanism that blocks replication from occurring twice in the same division cycle.

Histone Synthesis and Assembly

• Histones are coded for by multiple copies of the coding sequence.
• They are produced during S Phase, and not during other times of the cell cycle.
• The amount of histone protein is equal to the total mass of chromosomal DNA.
• The mRNAs breakdown rapidly after S Phase.
• How can different types of histones associate with the correct regions of DNA?
• Old come back together in the same region as they were prior to replication, and the old ones influence the modifications made to the new ones in the same area.
• Assembly occurs just after strand synthesis but the nucleosomes mature for an hour after synthesis.

Telomeres

• These are the short G-rich repeats found at the end of the linear chromosomes of eukaryotes.
• The repeated sequence of GGGTTA make up the human teleomeres.
• Telomerase is the enzyme that makes these.
• Telomerase has a bound RNA template that it uses over and over to create these repeats.
• It is a reverse transcriptase.