Replication


Creating the Pattern for DNA

Why DNA?

genotype: genetic information

phenotype: the observable traits or characteristics that result from the interaction of the genotype with the environment.

  • DNA can be copied quickly and accurately

  • can be passed down from generations (heritable)

DNA is double stranded, the nucleotide building blocks of DNA always match up in a complementary fashion

DNA runs antiparallel

*DNA replication is semi conservative. This means that half of the molecule is “conserved” or left in its original state

How DNA copies itself    

Key Players

  • Template DNA

  • Nucleotides

  • Enzymes and various proteins

Template DNA

  • Every organism’s DNA exists in the form of chromosomes. During DNA replication, cells use the chromosomes being copied as the pattern for making new chromosomes.

  • Both strands of each double'-stranded original molecule are copied, and therefore, each of the two strands serve as a template for replication

Nucleotides

The nucleotides building blocks that come together during replication are called deoxyribonucleic triphosphates (dNTPs)

  • Deoxyribose

  • One of the four bases

  • Three phosphates

The three phosphate groups are at the 5’ end of the molecule, and at the bottom of the molecule, AKA the 3’ spot, there is a little tail hydroxyl group that allows each nucleotide to hook up with DNA

When DNA is being replicated, the OH tail on the 3’ end of the last nucleotide in the chain reacts with the newly arrived dNTPs. Two of the phosphate groups get chopped off, and the remaining phosphate forms a phospodiester bond with the previously incorporated nucleotide

Enzymes

  • helicase: unzips the double helix so replication can occur

  • gyrase: prevents the helix from twisting too tightly and forming knots

  • primase: lay down a short piece of RNA primer to get replication started

  • DNA polymerase: adds dNTPs to build the new strand of DNA

  • ligase: seals the gaps between newly replicated pieces of DNA

  • telomerase: replicates the ends of the chromosomes (the telomeres)

Splitting the Helix

a. DNA replication begins at specific spots, called origins of replication, along the template DNA

b. Special proteins called initiators move along the double stranded template DNA until they encounter a group of bases that are in a specific order. Represent origin of replication. The proteins make a small opening in the double helix

  • Helicase finds this and begins breaking down hydrogen bonds between the base pairs

  • This unwinding creates two single-stranded DNA templates that allow for the synthesis of new complementary strands during replication.

c. DNA replication is bidirectional, meaning enzymes work in both directions AWAY from the origin

d. DNA has a tendency to form double strands if left alone, so after helicase does its job, proteins called single-stranded-binding proteins prop the two strands apart so that replication can occur

e. gyrase prevents DNA from being supercoiled by helicase split

  • in eukaryotes, gyrase is topoisomerases

Priming the process

  • Without a starting chain, the dNTPs cannot begin to link together, although there are two available single strands for replication

  • primase lays down RNA primers for replication at each replication fork so that DNA synthesis can begin on both ends. DNA uses the primer’s OH tails to add dNTPS; primers are later snipped out and replaced with DNA

  • This process is crucial because it provides the necessary 3' hydroxyl group that DNA polymerases require to initiate synthesis, ensuring the continuity of the newly synthesized strands.

    • in bacteria, DNA polymerase III builds the new partner strand of DNA—adds nucleotides to the growing strand by catalyzing the formation between 5’ end of an incoming dNTP and the reactive 3’ OH of the growing strand. Builds DNA in the 5’ → 3’ direction

In eukaryotes, the process is slightly more complex, involving multiple DNA polymerases, including DNA polymerase α, which synthesizes the RNA-DNA primers, and DNA polymerase δ, which carries out the elongation of the lagging strand. Additionally, the presence of multiple origins of replication allows eukaryotic cells to efficiently duplicate their larger genomes.

Leading and Lagging

  • At the OH end of each primer, DNA polymerase tacks on dNTPs by snipping off two phosphates and forming phosphodiester bonds to create the sugar-phosphate backbone of DNA.

    • Strand with primer from 3’→5’ is easy for DNA polymerase to add to: called the leading strand

    • Strand with primer from 5’→3’ requires more work; called the lagging strand

      • 3’ is pointing away from the replication fork; therefore, primase keeps putting down new primers as the crew moves along. Made in short pieces

  • DNA replication can only happen in the 5’ → 3’ direction

  • Leading strand: the strands formed in one bout of uninterrupted DNA synthesis. Leading strands follow the lead, so to speak, of helicase. 3’ end points towards replication fork

  • Lagging strands: strands that are begun over and over as new primers are laid down. Synthesis of the lagging strands stops when they reach the 5’ end of a primer elsewhere on the strand—replication happens so fast, there is not time difference.

  • The short pieces of DNA formed by lagging DNA synthesis are called Okazaki fragments

  • A special kind of DNA polymerase moves along the newly synthesized strands seeking out the RNA primers. When this DNA polymerase encounters the short bits of RNA, it snips them out and replaces them with DNA, follows same order as real replication

  • After the RNA primers are removed and replaced with DNA, one phosphate bond is missing between the Okazaki fragments. Ligase is the enzyme that seals these little gaps to complete the sugar-phopshate backbone.

*Prokaryotes have 5 forms of DNA polymerase, and eukaryotes have at least 13 forms.

Proofreading replication

  • speed: 2,000 bases per minute—bacterial is faster, at 1,000 bases per second

    • DNA polymerase makes a 1 out of every 100,000 bases mistake


DNA polymerase checks its work through a process called proofreading. If an incorrect base is added, DNA polymerase backs up and cuts the incorrect base out. The snipping process is called exonuclease activity, and the correction process requires polymerase to move in the opposite direction

  • error with proofreading is one out of 10 million pairs

If DNA polymerase misses an incorrect base, special enzymes come along after replication to complete another process, called mismatch repair. Detects bulges along the helix with noncomplementary bases are paired up.

  • Enzymes snip the incorrect base out of the newly synthesized strand

Replication Review

  • Replication always starts at an origin

  • Replication can only occur when template DNA is single-stranded

  • Replication always movs 5’→ 3’

  • Newly synthesized strands are exact complementary matches to template “old” strands

Replication in Eukaryotes

Telomerase

When linear chromosomes replicate, the ends of the chromosomes, called telomeres, present special challenges. These challenges are handled in different ways depending on what kind of cell division is taking place (mitosis or meiosis)

As a completion of replication for cells in mitosis, a short part of the telomere tip is left single-stranded and unreplicated. A special enzyme comes along and snips off this unreplicated part of the telomere.

  • Telomeres are long strands of non coding DNA, repeated sequence of TTAGGG. Regulation of gene expression

After many rounds of replication, all the non-coding, repetitive DNA at the ends of chromosomes are cut off, and actual genes are affected. Therefore, when the chromosome of a mitotic cell gets too short, the cell goes through apoptosis

If a cell is being divided as a part of meiosis, telomere snipping is not okay. The telomeres must be replicated completely so that perfectly complete, full sized chromosomes are passed down to offspring

Telomerase replicates telomeres. Telomerase carries a short piece of RNA that matches the repeated DNA sequence of telomeres. Then, uses its RNA as a template to extend the unreplicated DNA template until its long enough to be primed and replicated

Finishing the Job

Eukaryotic DNA is tightly wrapped around special proteins called histones to form structures called nucleosomes. This allows the enormous molecule to fit neatly into the nucleus.

During DNA replication, nucleosomes ahead of the moving replication fork must be disassembled, and then reassembled after the DNA is copied. A group of proteins called histone chaperones help facilitate this process.

As helicase opens the DNA molecule during replication, histone chaperone proteins help disassemble the original nucleosomes. Old and new histones used

How do circular DNAs replicate?

  • Circular DNAs are replicated in three different ways

Theta

Refers to the shape the chromosome takes on during replication process. After the helix splits apart, a bubble forms, giving the chromosome a shape like the greek later theta. Bacterial replication only has one origin, DNA replication proceeds normally

Rolling Circle

No primer is needed because the double stranded template is broken at the origin to provide a free OH tail to start replication. As replication proceeds, the inner strand is copied continuously as the leading strand. Broken strand is stripped off. As soon as enough of the broken strand is free, a primer is laid down so replication can occur as the broken strands is stripped away from its complement

Viral DNA

D-loop

Creates displaced, single strand. Helicase opens up the double stranded molecule, and an RNA primer is laid down, displacing one strand. Replication then proceeds around the circle, pushing the displaced strand off as it goes. The intact, single strand is released and used as a template to synthesize a complementary strand.