DNA Structure and Replication: Study Notes for Genetics Students. CH 7

DNA Structure and Replication

DNA as the Hereditary Molecule

DNA is the fundamental hereditary molecule in all living organisms, responsible for storing and transmitting genetic information. Its identification as the hereditary material was based on several key characteristics:

• Localization: DNA is found in the nucleus and is a component of chromosomes.

• Stability: DNA exists in a stable form within cells.

• Complexity: DNA is sufficiently complex to encode the information required for structure, function, development, and reproduction.

• Replication: DNA can accurately replicate itself, ensuring daughter cells inherit identical genetic information.

• Mutability: DNA undergoes mutations at a low rate, providing genetic variation essential for evolution.

Historical Experiments Identifying DNA as Hereditary Material

• Griffith's Transformation Experiment: Demonstrated that a 'transformation factor' could transfer genetic traits between bacterial strains, suggesting the existence of a hereditary molecule.

• Avery, MacLeod, and McCarty: Showed that DNA, not protein or RNA, was responsible for transformation in bacteria, confirming DNA as the hereditary molecule.

• Hershey and Chase: Used radioactive labeling to demonstrate that DNA, not protein, enters bacteria during phage infection, further confirming DNA's role in heredity.

The DNA Double Helix

The secondary structure of DNA, elucidated by Franklin, Watson, and Crick, is a double helix composed of two complementary and antiparallel strands. Each strand is a polymer of nucleotides joined by phosphodiester bonds.

DNA Nucleotides

• Components: Each nucleotide consists of a deoxyribose sugar, a nitrogenous base (adenine, thymine, cytosine, or guanine), and one to three phosphate groups.

• Base Classification: Pyrimidines (thymine, cytosine) have a single ring; purines (adenine, guanine) have a double ring.

• Polymerization: DNA polymerase catalyzes the formation of phosphodiester bonds between the 3' hydroxyl group of one nucleotide and the 5' phosphate of the next, forming a sugar-phosphate backbone.

Complementary and Antiparallel Strands

• Complementarity: Adenine pairs with thymine (A-T) via two hydrogen bonds; guanine pairs with cytosine (G-C) via three hydrogen bonds.

• Antiparallel Orientation: The two strands run in opposite directions (5' to 3' and 3' to 5').

Structural Features of the Double Helix

• Helical Structure: The B-form DNA has a right-handed twist, a diameter of 20 Å, and base pairs spaced at 3.4 Å intervals.

• Major and Minor Grooves: Base-pair stacking creates alternating major (12 Å) and minor (6 Å) grooves, which are important for protein binding.

Forms of DNA

DNA can exist in three structural forms: A-form, B-form, and Z-form, each with distinct physical properties.

Form	Helical Twist	Rotation per Base Pair	Base-Pair Spacing	Base Pairs per Turn	Helix Diameter
A-Form	Right-handed	32.7°	2.6 Å	11	23 Å
B-Form	Right-handed	34.3°	3.4 Å	10.5	20 Å
Z-Form	Left-handed	60.2°	3.7 Å	12	18 Å

DNA Replication

DNA replication is a highly accurate and conserved process, ensuring genetic continuity across generations. It is semiconservative and bidirectional in most organisms.

Models of DNA Replication

• Semiconservative: Each daughter duplex contains one parental and one newly synthesized strand.

• Conservative: One duplex contains both parental strands; the other contains both daughter strands.

• Dispersive: Each duplex contains interspersed segments of parental and daughter DNA.

Origin and Directionality of Replication

• Bacterial Chromosomes: Replication initiates at a single origin and proceeds bidirectionally, forming a replication bubble and forks.

• Replication Bubble: Electron micrographs reveal the replication bubble and forks.

• Eukaryotic Chromosomes: Multiple origins of replication are present, as seen in Drosophila chromosomes.

Eukaryote Replication Origins

• Yeast: Origins are called autonomously replicating sequences (ARS), with well-characterized consensus sequences.

Molecular Biology of Replication Initiation

• Bacteria: Enzymes DnaA, DnaB, and DnaC bind to oriC to initiate replication.

• Archaea: Orc1/Cdc6 complex binds to ORB and miniORB sequences.

• Eukaryotes: A prereplication complex with 14 proteins, including the origin replication complex (ORC), assembles at origins.

• DNA Polymerase: Elongates DNA by adding nucleotides to the 3' end; requires RNA primers synthesized by primase.

Continuous and Discontinuous Strand Replication

• Leading Strand: Synthesized continuously in the direction of fork progression.

• Lagging Strand: Synthesized discontinuously via Okazaki fragments, in the opposite direction.

RNA Primer Removal and Okazaki Fragment Ligation

• DNA Polymerase I: Removes RNA primers (5'-3' exonuclease) and replaces them with DNA (5'-3' polymerase).

• DNA Ligase: Seals gaps between DNA fragments, forming a continuous strand.

DNA Proofreading

• Accuracy: DNA polymerases possess 3'-5' exonuclease activity for proofreading, removing mismatched nucleotides and correcting errors.

• Error Rate: Errors occur approximately once per billion nucleotides in E. coli.

Replication at the Ends of Linear Chromosomes

• Telomeres: Repetitive sequences at chromosome ends protect against loss of genetic material during replication.

• Lagging Strand Problem: The requirement for a primer prevents complete replication of the lagging strand, leading to telomere shortening.

Telomerase

• Function: Telomerase is a ribonucleoprotein that extends telomeres in germ line and certain other cells, using its RNA template to add DNA repeats.

• Significance: Telomere length is crucial for chromosome stability, cell longevity, and reproductive success. Activation of telomerase is observed in most cancer cells.

Methods of Molecular Genetic Analysis

DNA replication processes are utilized in molecular genetic techniques such as polymerase chain reaction (PCR) and dideoxynucleotide DNA sequencing.

VNTR Analysis via PCR

• VNTRs: Variable Number Tandem Repeats are short, repetitive DNA sequences that vary in number between individuals.

• PCR: Amplifies VNTR regions, producing fragments of different lengths based on repeat number.

• Band Patterns: Gel electrophoresis separates PCR products, revealing VNTR band patterns for genotyping.

• Inheritance: VNTR variation can be tracked through pedigrees, demonstrating Mendelian inheritance.

Additional info: The notes expand on brief points with academic context, definitions, and examples to ensure completeness and clarity for Genetics students.