Lecture 12: Genetic Material and DNA Replication

Number of Chromosomes in Human Cell

• 23 PAIRS of chromosomes

  • 46 TOTAL

  • CONSIST of double-stranded DNA

Composition of Chromosomes

• POLYMER (DNA STRAND)

  • Millions of nucleotides

• TWO complementary DNA strands

  • HELD via hydrogen bonds

Do Organisms Have Same Number of Chromosomes

• NO

• Different organisms have DIFFERENT numbers of chromosomes

  • EXAMPLE: Bacteria → one chromosome

Number of Genes in Each Chromosome

• HUNDREDS to THOUSANDS

What Each Gene Encodes

• EACH gene encodes ONE protein

• HUNDREDS to THOUSANDS of nucleotides make up each gene

Central Dogma Definition

• DNA → RNA → Protein

• Passes Information

Central Dogma Brief Steps

1. Genes are TRANSCRIBED to RNA

2. RNA is TRANSLATED to protein

3. Proteins do WORK in cell

Are Genes Transcribed at Once?

• NOT ALL genes transcribed at once

Why All Genes Are Transcribed at Once

• WHEN and WHERE proteins made DETERMINES cell function

Genome

• HOLDS genetic information

DNA

• GENETIC MATERIAL → For all living cells on planet

• MOSTLY exist in chromosomes → MUST be passed from parent to daughter cell

DNA is Heritable

• Genetic material PASSED from parent to offspring

DNA and Phenotypes

• DETERMINES characteristics of organism

Evidence for DNA as Genetic Material

• TRANSFORMATION OF STREPTOCOCCUS (Griffith’s Experiment)

Griffith Experiment Goal

• DETERMINE how phenotypes can be passed from one cell to another

  • ALLOWS for assumption of GENETIC MATERIAL

Streptococcus

• Pathogenic bacteria

• Can cause disease

Streptococcus Forms

• Pathogenic

• Non-pathogenic

Pathogenic Streptococcus

• S STRAIN

• SMOOTH and SHINY colonies

Non-Pathogenic Streptococcus

• R STRAIN

• ROUGH and DRY colonies

• Benign (not harmful) form

Dead S (pathogenic) Bacteria and Living R (non-pathogenic) Bacteria

• Dead S (pathogenic) bacteria contains substance

• TRANSFORMS R (non-pathogenic) bacteria into pathogenic S strain

Transforming Factor

• SOME MOLECULE in pathogenic Streptococcus

• Could TRANSFORM rough (non-pathogenic) bacteria into smooth (pathogenic) bacteria

Avery, McCarty, and Macleod Experiment Setup

• START with heat-killed Streptococcus

  • CONTAINS different macromolecule types → Lipid, Carbohydrates, Protein, RNA, DNA

• REMOVE lipids and carbohydrates

• ADD enzyme (to digest one macromolecule type) in treatment

• ADD each treatment to R (non-pathogenic) Cell mixture

Avery, McCarty, and Macleod Experiment Results

• DNA is TRANSFORMING FACTOR (non pathogenic to pathogenic bacteria)

  • RNA + DNA → Transformation

  • Protein + DNA → Transformation

  • Protein + RNA → No Transformation

Macromolecule That is Transforming Factor

• DNA

DNase

• GROUP of enzymes

• ELIMINATES transformation ability

Purified DNA

• In S (pathogenic) strain

• Can TRANSFORM to R (non-pathogenic) strain

How Genetic Information is Passed

• FROM parent to offspring (one cell to another)

• VIA cell division

When and Why DNA Replication

• BEFORE cell division

• EACH daughter recieves exact copy of genetic material

Watson-Crick Model Implications

• INFORMATION in nitrogenous base sequence

• STRUCTURE OF DNA suggest possible copying mechanism

DNA Replication Start

• BREAKS hydrogen bonds between complementary base pairs

• SEPERATES DNA helix

DNA Strands During Replication

• EACH original strand becomes template

  • FOR new complementary strand

Free Nucleotides and New DNA Strand

• Free nucleotides PAIR with complementary bases

• POLYMERIZE to form sugar-phosphate backbone

Result of DNA Replication

• Two IDENTICAL DNA molecules

  • One NEW strand

  • One ORIGINAL strand

Restoring Secondary Structure of DNA After Replication

• New strands POLYMERIZE to form sugar-phosphate backbone

• RESTORES secondary structure

Bonds Between Nucleotides During DNA Replication

• Phosphodiester bonds

DNA Replication Guide

• Template Strand

DNA Replication Building Blocks

• Deoxyribonucelotide triphosphates (dNTPs)

DNA Replication Catalyst

• DNA polymerase

  • Enzyme

Direction of New Strand Synthesis

• 5’ to 3’

Energy to Form a Phosphodiester Bond

• Phosphate group

  • FROM incoming nucleotide

New Nucleotides in DNA Replication

• ADDED to existing chain

• MAKES phosphodiester bonds

DNA Polymerase Requirements

• Template strand

• Primer

  • 3’ OH

  • FREE 3’ group

• dNTPs

DNA Polymerase Characteristic

• VERY fast

• VERY accurate

Direction of RNA Replication in Linear Chromosome

• Bidirectional

Type of DNA Eukaryotic Chromosomes

• Linear

• Double-Stranded

Each Parental Strand During DNA

• SERVE as template for new strand

Chromosome Replication Start Location

• Origin

Replication Bubble

• Region

• Two strands SEPARATE at origin

Direction of Replication Forks

• AWAY from origin of replication

• BOTH directions

RNA Primer Functions in DNA Replication

• STARTING point

• FOR DNA synthesis

Leading Strand

• CONTINUAL synthesis of new DNA strand

• 5’ to 3’ direction

• TOWARDS replication fork

Lagging Strand

• DISCONTINUOUS synthesis of new DNA strand

• 5’ to 3’ direction

• Short Okazaki fragments

Okazaki Fragments

• SHORT DNA segments

• SYNTHESIZED on lagging strand

Result of Bidirectional DNA Replication

• TWO IDENTICAL double stranded DNA molecules

  • One PARENTAL strand

  • One NEW strand

Leading Strand Synthesis Steps

1. DNA opened, unwound, and primed

2. Synthesis of leading strand begins

Primer

• SHORT stretch of nucleic acid

• MAKES base pairs with template strand

• PROVIDES 3’OH for DNA polymerase

  • TO use and add new dNTP starting DNA synthesis

Primase

• Enzyme

• MAKES RNA primer during DNA replication

DNA/RNA Synthesis Direction

• ALWAYS in 5’ to 3’ direction

  • FIRST NUCLEOTIDE ADDED → 5’ end

Topiosomerase

• RELIEVES DNA twisting force

Helicase

• OPENS double helix

• BREAKS hydrogen bonds between bases

Single-Stranded DNA-Binding Proteins (SSBP)

• Stabilizes single strands

DNA Polymerase Direction

• 5’ to 3’ direction

• SYNTHESIZES leading strand

Synthesizing DNA Strand Continuously and Non-Continuously Reason

• DNA strands are:

  • Antiparallel

  • Synthesize in one direction

Lagging Strand Synthesis Steps

1. PRIMASE synthesizes RNA primer

2. DNA POLYMERASE synthesizes Okazaki fragments in 5’ → 3’ direction

3. PRIMASE AND DNA POLYMERASE repeat process to synthesize additional Okazaki fragment

4. DNA POLYMERASE removes RNA primers and replaces with DNA nucleotide (5’ → 3’ direction)

5. DNA ligase seal gap in sugar-phosphate backbone, joining fragments together

Reason Replicating Ends of Linear Chromosomes Becomes a Problem

• Last RNA primer on lagging strand REMOVED

• LEAVES gap at end of DNA

Result of Removing the Final RNA Primer from Lagging Strand

• Gap CANNOT be filled

• SOME DNA at chromosome lost during subsequent replication

Telomerase in DNA Replication

• ENZYME

• EXTENDS 3’ end of DNA

• ALLOWS new primer to be added so DNA synthesis can occur

Reasons Organisms with Linear Chromosomes Need Telomerase

• PREVENT chromosomes from shortening after each round of DNA replication

• BOTH ends of chromosome

Telomere Replication Process

1. End is unreplicated

2. Telomerase extends unreplicated end (does it again on other strand)

3. Lagging Strand is complete