Biology Chapter on DNA Replication Mechanics

DNA Replication Overview

• Base-pairing enables DNA replication
• DNA synthesis starts at replication origins
• DNA replicates semi-conservatively
• DNA replication process details
• Telomeres and telomerase function

DNA Replication Details

• Cell survival and proliferation depend on accurate genetic duplication
• DNA replication happens before cell division to create two identical daughter cells
• DNA replication happens during the synthesis phase
• Each DNA strand is a template for a complementary strand

Base-Pairing Enables DNA Replication

• Each strand of a DNA double helix has a nucleotide sequence complementary to its partner strand
• Each strand acts as a template to produce a complementary strand

DNA Copying Mechanisms

• Three models of DNA replication exist (Watson and Crick, Delbrück, Model #3)
• Semiconservative: After replication, the new DNA molecule consists of one original (parental) strand and one newly synthesized strand
• Dispersive: During replication, segments of parental strand mix with newly synthesized DNA
• Conservative: Two original parental DNA strands and two newly synthesized DNA strands are produced

Labelling DNA with Stable Isotopes

• Meselson and Stahl used E. coli, heavy 15N-containing DNA and light 14N-containing DNA
• Centrifugation in CsCl gradient separates heavy and light DNA
• Equilibrium density centrifugation was used

Testing DNA Replication Hypotheses

• Various conditions and results were tested to identify the correct replication model.
• Results from various methods support the semiconservative model

DNA Synthesis Begins at Replication Origins

• DNA double helix is usually stable
• Opening the helix requires energy, like heat
• Initiator proteins bind to specific replication origins.
• Replication origins are locations in the genome where replication begins
• Bacteria have circular chromosomes with 1 origin
• Humans have linear chromosomes with many origins ( ~220 per chromosome/ ~10,000 total)

Two Replication Forks Form at Each Replication Origin

• DNA synthesis occurs at Y-shaped junctions called replication forks
• Two forks are produced at the origin
• At each fork, the replication machine moves along the DNA, using each strand as a template to make a new daughter strand.
• Replication is bidirectional
• Speed = ~1,000 nt/sec (bacteria) and ~100 nt/sec (humans)

DNA Polymerase Synthesizes DNA Using a Parent Strand as a Template

• The replication machine uses DNA polymerase
• DNA polymerase uses one parent DNA strand as a template
• Catalyses nucleotide additions at the 3' end of the growing DNA strand
• DNA is synthesized in a 5' to 3' direction

DNA Polymerase Adds Deoxyribonucleotide to the 3' end of a Growing DNA Strand

• Formation of phosphodiester bonds joining the 3' end of the growing chain to the 5' phosphate group of the incoming nucleotide
• Pyrophosphate is hydrolyzed, making the polymerization reaction irreversible

DNA Polymerase Proofreads its Own Work During DNA Synthesis

• DNA polymerase has high accuracy (~1 error in 10⁷ nucleotides)
• Monitoring base pairing between incoming nucleotide and template helps ensure accuracy
• When a mistake occurs, the polymerase corrects it through proofreading

Initiation of DNA Synthesis Requires

• DNA polymerase needs a base-paired 3' end before adding nucleotides.
• A separate enzyme, primase, provides this starting point.
• Primase synthesizes RNA primers for DNA replication

Multiple Enzymes Required for Discontinuous DNA Synthesis

• New RNA primers are laid down at intervals along the lagging strand.
• DNA polymerase adds deoxyribonucleotides to the 3' end of each primer to form Okazaki fragments
• Three other enzymes are needed to generate a continuous new DNA strand from Okazaki fragments

DNA Ligase

• Joins the 5' phosphate end of one fragment to the 3' hydroxyl end of the next
• Joins Okazaki fragments on the lagging strand

Proteins at Replication Fork Cooperate to Form a Replication Machine

• DNA replication requires the double helix to be continually separated by DNA helicase (ATP dependent)
• Single-strand DNA binding proteins (SSBs) latch onto ssDNA to prevent re-annealing

Replication Machine

• DNA synthesis is carried out by a group of proteins.
• DNA polymerase operates in the 5' to 3' direction.
• Sliding clamp to keep DNA polymerase attached to the template.
• Clamp loader assists in locking the sliding clamp onto the DNA.

Replication Machine in Cells

• Proteins in the replication machine are held together in a large unit
• Clamp loader locks the sliding clamp around the newly formed DNA template
• ATP is required in this process

Does Localized Unwinding of DNA Double Helices Present a Problem?

• As helicase moves, the DNA ahead of the replication fork tightens.
• Tension builds up, but this problem is relieved by DNA topoisomerases

DNA Topoisomerases

• Enzymes that relieve tension by making transient single-strand breaks in the DNA.
• Breaks are resealed to release tension.

Various Proteins and their Activities in DNA Replication

• Summary of various proteins and their roles in DNA replication

DNA Replication in Living Cells

• The given website is a resource for advanced information on DNA replication

Telomerase Replicates the Ends of Eukaryotic Chromosomes

• Leading strand replication is completed but lagging strand replication cannot
• Telomeres are required to finish lagging strand replication.
• RNA primer is removed and replaced by DNA using a mechanism of proofreading
• Telomerase carries its own RNA template and uses it add copies to the lagging strand template

Dealing with "End-Replication Problem"

• Telomeres are repetitive nucleotide sequences at the ends of chromosomes
• Provide extra DNA for lagging-strand replication completion
• Telomerase carries its own RNA template, adding repetitive DNA sequences to the lagging strand template

Telomeres and Telomerases

• Telomeres prevent linear eukaryotic chromosomes from shortening after each cell division
• Repetitive DNA sequences draw telomere-binding proteins to protect chromosome ends and maintain telomere length

Telomeres and Telomerases

• Telomere length varies with age and cell type.
• Rapidly dividing cells maintain high telomerase activity, whereas many other cell types gradually reduce their telomerase activity over cell divisions.
• Telomeres shorten, eventually causing cell division to cease.

Lecture 3 Reading

• Chapter 6 covers DNA replication and repair, on pages 179-225 of the textbook

Upcoming Lecture 4 Reading

• Chapter 6 covers DNA replication and repair, on pages 225-236

Room Changes for Labs 55 and 60

• Room changes for labs 55 and 60 are to be found on UWinsite