DNA Replication: Process and Models

DNA Replication Overview

• DNA replication is vital for cell replication, ensuring each new cell gets a complete set of genetic information.
• The main purpose of DNA replication is to help cell replication, which lets the cell cycle move forward and make new cells.
• DNA replication mainly happens during the S phase of the cell cycle.
• During cell replication, a cell copies its DNA, which includes 23 maternal and 23 paternal chromosomes, to make two identical daughter cells.

Semi-Conservative Model

• DNA replication uses a semi-conservative model, where each new DNA molecule has one original and one newly made strand.
• In this model, the two DNA strands split, and each acts as a template for a new complementary strand.
• The original strands are called "old" or parental strands, while the new ones are "new" or daughter strands.

Direction of Replication

• DNA replication goes in a specific direction, always from the 5' (phosphate group) end to the 3' (OH group) end.
• Nucleotides are added by connecting a phosphate group (5' end) to the OH group (3' end) of the nucleotide before it.
• DNA strands are antiparallel, meaning one goes from 5' to 3', and the other goes from 3' to 5'.
• When copying a DNA strand, the new strand is made in the 5' to 3' direction, matching the template strand.

Bi-Directional Replication

• DNA replication is bi-directional, moving in both directions from the origin of replication.
• When the two parental strands separate, it creates Y-shaped areas called replication forks.
• Helicases unwind the DNA at these forks, moving in both directions.
• DNA polymerases then follow the helicases, making new DNA strands in both directions.

Steps of DNA Replication

• DNA replication has three main steps: initiation, elongation, and termination.

Initiation

• Initiation starts at specific areas on the DNA called origins of replication.
• These origins have lots of adenine (A) and thymine (T) nucleotides, which are easier to separate because they only have two hydrogen bonds, unlike guanine (G) and cytosine (C) pairs that have three.
• Eukaryotic cells have several origins of replication to speed up the copying process.
• The pre-replication protein complex binds to the origin of replication, separating the DNA strands and forming a replication bubble.
• Single-stranded binding proteins attach to the separated parental DNA strands to keep them from re-annealing, or reconnecting.
• These proteins also protect the single strands from being broken down by nucleases.
• Replication forks form at the ends of the replication bubble, creating a Y-shaped shape.
• Helicase, an enzyme needing ATP, unwinds the DNA at both replication forks.

Supercoils and Topoisomerases

• As helicase unwinds the DNA, it causes the DNA ahead of the replication fork to bunch up, forming supercoils.
• Supercoils slow down helicase, making it harder to unwind the DNA.
• Topoisomerases are enzymes that fix supercoils by cutting, untwisting, and rejoining DNA strands.
• There are different types of topoisomerases like type 1, type 2, and type 4.

Topoisomerases

• These enzymes relieve DNA supercoils by cutting the DNA strand to allow unwinding.
• They have two domains: nuclease and ligase.
• The nuclease domain cuts or breaks the phosphodiester bond in one or two DNA strands, allowing unwinding.
• The ligase domain re-stitches the DNA after the supercoils are relieved.
• Eukaryotic cells mainly have type 1 and 2 topoisomerases, while prokaryotic cells mostly have types 2 and 4.
• Type 1 topoisomerase does not need ATP to unwind supercoils.
• Types 2 and 4 need ATP to unwind supercoils.
• Types 2 and 4 can cut supercoiled DNA, allow unwinding, and insert negative supercoils to relax the DNA.
• Topoisomerases are drug targets for cancer and bacterial infections.

Clinical Applications and Drugs

• Targeting topoisomerases in eukaryotic cancer cells can prevent replication.
• Cancer drugs for topoisomerase 1 include irinotecan and topotecan.
• Drugs for topoisomerase 2 in eukaryotic cells: etoposide and teniposide.
• Targeting topoisomerases 2 and 4 in prokaryotic cells can prevent bacterial replication in bacterial infections.
• Fluoroquinolones (e.g., ciprofloxacin, levofloxacin) inhibit topoisomerase 2 in bacteria.
• These drugs increase the activity of the nuclease domain while inhibiting the ligase domain, leading to DNA fragmentation.

DNA Elongation

• After unwinding and stabilizing the DNA, the next step is elongation.
• Primase makes RNA primers, which allow DNA polymerase type 3 to make DNA.
• Primase reads the DNA strand from 3' to 5' and makes RNA primers in the 5' to 3' direction.
• DNA polymerase type 3 needs the 3' OH of the RNA primer to build nucleotides.
• DNA polymerase type 3 reads the DNA from 3' to 5' and makes nucleotides in the direction 5' to 3'.
• The leading strand is continuous, needing only one RNA primer.
• DNA polymerase moves towards the replication fork on the leading strand.
• The lagging strand needs multiple RNA primers, made by primase.
• Okazaki fragments are sections of DNA between RNA primers on the lagging strand.

Addressing RNA Primers and Proofreading

• RNA primers need removal because replicated DNA should be all DNA.
• Before removing RNA primers, understand the proofreading function of DNA polymerase type 3.
• DNA polymerase type 3 proofreads to prevent mistakes.
• It reads from 3' to 5' and checks for correct complementary base pairs.
• If there’s a mistake, it uses 3' to 5' exonuclease activity to cut out the wrong nucleotide.
• Then, it synthesizes the correct nucleotide in the 5' to 3' direction.
• DNA polymerase type 1 removes the RNA primers.

DNA Polymerase I Functions

• DNA Polymerase I locates the primers.
• It removes primers using 5' to 3' exonuclease activity, specifically plucking out the RNA primers.
• After plucking out the primers, it reads the DNA strand from 3' to 5'.
• It synthesizes a new strand from 5' to 3' based on the template strand it read.
• It proofreads its work.
• If it finds an incorrect base pair connection, it cuts it out in a 3' to 5' exonuclease fashion.

Comparison with DNA Polymerase III

• DNA Polymerase I can do everything DNA Polymerase III does.
• DNA Polymerase I also has 5' to 3' exonuclease activity for removing RNA primers.

Activity on Lagging Strand

• On the lagging strand, DNA Polymerase I uses its 5' to 3' exonuclease activity.
• It removes the RNA primers on the lagging strand.
• It synthesizes new DNA in the 5' to 3' direction to fill the gaps left by the removed primers.

DNA Polymerase Action

• DNA polymerase moves along the DNA, removing RNA primers.
• It reads DNA in the 3' to 5' direction.
• It synthesizes new DNA in the 5' to 3' direction.
• Proofreads the newly synthesized DNA to ensure accuracy.
• If an error is found, it uses its 3' to 5' exonuclease activity to remove the incorrect nucleotide and inserts the correct one.
• On the lagging strand, DNA polymerase creates gaps between Okazaki fragments where RNA primers were located.
• These gaps mean that the DNA cannot completely fuse.

Ligase Function

• Ligase is an enzyme that fuses the DNA ends together on the lagging strand.
• It connects Okazaki fragments to create a continuous DNA strand.
• Ligase ensures that the new daughter DNA strand is perfectly connected, continuous, and in sequence.
• The final product is a parental DNA strand with a new, continuous daughter DNA strand.

Clinical Significance: HIV and Nucleoside Reverse Transcriptase Inhibitors (NRTIs)

• HIV infects T cells, incorporating the viral genome into the T cell's DNA.
• When these T cells replicate, they also replicate the HIV genome.
• NRTIs are drugs used to target the HIV virus and inhibit replication in infected T cells.
• NRTIs are nucleoside analogs that interfere with the DNA replication process.
• DNA polymerase III needs a 3' OH region to build the DNA strand.
• NRTIs lack the 3' OH region, so when incorporated into the growing DNA strand, they stop further elongation.
• NRTI incorporation inhibits complete DNA replication in affected T cells.
• Examples of NRTIs include didanosine and zidovudine.

Termination of DNA Replication

• Termination happens when DNA polymerases moving towards each other at replication forks meet.
• Helicases unwind the DNA, and when they meet, they stop unwinding.
• The DNA polymerases then detach from the DNA.
• DNA replication starts at multiple origins and goes bidirectionally until the replication forks meet.

Telomeres and Telomerase

• Telomeres are at the ends of chromosomes.
• Telomeres get shorter over time as cells replicate.
• Chromosomes have two structural key points: centromeres and telomeres.
• Telomeres do not code for RNA or any proteins.
• Telomeres sacrifice themselves during DNA replication to prevent gene loss.
• The Hayflick limit is the maximum number of times a cell can replicate before telomere shortening interferes with genes.
• On the lagging strand, DNA polymerase I removes RNA primers but cannot add nucleotides without a 3' OH end.
• This shortens telomeres with each replication cycle, so the new strand is shorter than the last one.
• Telomerase is a special enzyme that lengthens telomeres.
• Telomerase is a ribonucleoprotein with two arms.
• Telomerase expresses complementary nucleotides commonly found on telomeres (TTAGGG).
• Telomerase uses its RNA template to synthesize DNA (reverse transcription).
• It elongates the 3' end of the parental DNA strand.
• Telomerase prevents significant telomere shortening and gene loss.
• Telomerase is highly active in stem cells and highly replicating cells.
• Cancer cells can increase telomerase activity, allowing them to replicate forever.
• Telomerase performs reverse transcription (RNA to DNA).
• Telomerase uses one arm with RNA to build DNA on the parental strand, elongating it.