DNA Replication, Recombination, and Chromatin: Structure and Function in Prokaryotes and Eukaryotes

Prokaryotic and Eukaryotic Genomes

Prokaryotic Genome Organization

Prokaryotic genomes are typically compact and organized in a circular structure, with essential genetic information required for cell survival and replication.

• Chromosomal Structure: Most bacterial species contain a single, circular chromosomal DNA molecule. However, some species may have more than one chromosome or linear chromosomes (rare).

• Plasmids: In addition to the main chromosome, bacteria often carry one or more small, circular DNA molecules called plasmids, which can carry genes beneficial for survival (e.g., antibiotic resistance).

• Gene Content: A typical bacterial chromosome contains several thousand genes, interspersed with short repetitive sequences.

• Origin of Replication: There is usually a single origin of replication (oriC) per chromosome, which is the site where DNA replication is initiated.

Example: Escherichia coli has a single circular chromosome of about 4.6 million base pairs and several plasmids.

Eukaryotic Chromosome Organization

Eukaryotic genomes are more complex, with linear chromosomes and specialized structures for replication and segregation.

• Chromosomal Structure: Eukaryotic chromosomes are linear and much larger than prokaryotic chromosomes, ranging from tens of millions to hundreds of millions of base pairs.

• Gene Distribution: Genes are interspersed throughout the chromosome, separated by non-coding regions and repetitive sequences.

• Multiple Origins of Replication: Each chromosome contains many origins of replication, spaced approximately every 100,000 base pairs, to ensure timely duplication of the large genome.

• Centromeres and Telomeres: Each chromosome has a centromere (for kinetochore attachment during cell division) and telomeres (specialized repetitive sequences at chromosome ends for stability).

• Ploidy Levels: Human somatic cells are diploid (46 chromosomes), gametes are haploid (23 chromosomes), and mitochondria contain their own small circular chromosome.

Example: Human chromosome 1 is about 249 million base pairs long and contains over 2,000 genes.

Central Dogma and Information Flow

DNA as the Cell's Hard Drive

DNA stores genetic information, which must be faithfully copied and transmitted during cell division. The flow of genetic information is described by the central dogma of molecular biology.

• Replication: The process by which DNA is copied to produce identical DNA molecules for daughter cells. Catalyzed by DNA-dependent DNA polymerase.

• Transcription: The synthesis of RNA from a DNA template, catalyzed by DNA-dependent RNA polymerase.

• Reverse Transcription: The synthesis of DNA from an RNA template, catalyzed by RNA-dependent DNA polymerase (reverse transcriptase).

• Translation: The process by which mRNA is decoded by ribosomes to synthesize polypeptides (proteins).

Equation:

$\text{DNA} \xrightarrow{\text{Replication}} \text{DNA} \xrightarrow{\text{Transcription}} \text{RNA} \xrightarrow{\text{Translation}} \text{Protein}$

DNA Replication Mechanisms

Complementarity and Template FunctionxThe two strands of DNA are complementary, allowing each strand to serve as a template for the synthesis of a new strand during replication.

• Base Pairing: Adenine pairs with thymine, and guanine pairs with cytosine, ensuring accurate copying.

Models of DNA Replication

Three theoretical models were proposed for DNA replication:

• Conservative: The parental double helix remains intact, and an entirely new double helix is synthesized.

• Semiconservative: Each daughter DNA molecule consists of one parental (old) strand and one newly synthesized strand. This is the correct model.

• Dispersive: Parental and new DNA segments are interspersed in both strands after replication.

Initiation and Progression of Replication

• Origin of Replication: Replication begins at specific sites called origins of replication.

• Replication Fork: The site where the DNA double helix is unwound to allow synthesis of new strands.

• Bidirectional Replication: Replication proceeds in both directions from the origin, forming two replication forks.

Enzymes and Proteins in DNA Replication (Bacteria)

DNA replication in bacteria involves several key enzymes and proteins:

• DNA Polymerases: Enzymes that synthesize new DNA strands by adding nucleotides to a primer.

• Primase: Synthesizes short RNA primers required for DNA polymerase to begin synthesis.

• Helicase: Unwinds the DNA double helix at the replication fork.

• Single-Stranded Binding Proteins (SSBPs): Stabilize unwound DNA strands.

• Topoisomerase (including DNA gyrase): Relieves supercoiling ahead of the replication fork.

• Ligase: Seals nicks between adjacent DNA fragments.

Properties of Bacterial DNA Polymerases

Property	DNA Pol I	DNA Pol II	DNA Pol III
5'→3' Polymerization	Yes	Yes	Yes
3'→5' Exonuclease (Proofreading)	Yes	Yes	Yes
5'→3' Exonuclease	Yes	No	No
Molecules per cell	400	2	10-20

Additional info: DNA Pol III is the main replicative polymerase; DNA Pol I removes RNA primers and fills gaps.

Leading and Lagging Strand Synthesis

• Leading Strand: Synthesized continuously in the 5'→3' direction toward the replication fork.

• Lagging Strand: Synthesized discontinuously as short Okazaki fragments, each initiated by an RNA primer.

• DNA Ligase: Joins Okazaki fragments to form a continuous strand.

Key Steps in Bacterial DNA Replication

1. Unwinding of the helix (helicase, SSBPs)

2. Relief of supercoiling (topoisomerase/gyrase)

3. Synthesis of RNA primers (primase)

4. Elongation of new DNA strands (DNA Pol III)

5. Removal of RNA primers and gap filling (DNA Pol I)

6. Joining of DNA fragments (ligase)

Unique Features of Eukaryotic DNA Replication

• Multiple origins of replication per chromosome

• Linear chromosomes with telomeres

• DNA is complexed with histone proteins, forming chromatin

• Replication machinery is more complex, involving multiple DNA polymerases

DNA Recombination

Homologous Recombination

DNA recombination is a process by which genetic material is exchanged between similar or identical DNA molecules, increasing genetic diversity and aiding in DNA repair.

• Key Steps: Nicking of DNA, strand displacement and pairing, branch migration, and resolution of the Holliday junction.

• Enzymes: Specific enzymes direct the process, ensuring accurate exchange at homologous regions.

Example: Crossing over during meiosis in eukaryotes.

Chromatin Structure and DNA Packaging

Supercoiling and DNA Compaction

DNA supercoiling refers to the over- or under-winding of the DNA double helix, which helps compact the DNA to fit within the cell.

• Positive Supercoiling: Overwinding of the DNA helix, making it tighter.

• Negative Supercoiling: Underwinding of the DNA helix, making it looser and more accessible for processes like replication and transcription.

• Topoisomerases: Enzymes (including DNA gyrase) that introduce or remove supercoils in DNA.

Chromatin and Nucleosomes

In eukaryotes, DNA is packaged with proteins to form chromatin, which can exist in various levels of condensation.

• Nucleosome: The basic unit of chromatin, consisting of DNA wrapped around a core of histone proteins.

• Histones: Positively charged proteins (H1, H2A, H2B, H3, H4) that facilitate DNA compaction.

Histone Type	Lysine-Arginine Content	Molecular Weight
H1	Lysine-rich	23,000
H2A	Slightly lysine-rich	14,000
H2B	Slightly lysine-rich	13,800
H3	Arginine-rich	15,300
H4	Arginine-rich	11,300

Additional info: Nucleosomes are arranged in a linear array, further compacted into higher-order structures during cell division.

Chromatin Remodeling

Chromatin structure must be dynamically altered (remodeled) to allow access to DNA for replication, repair, and gene expression.

• Remodeling exposes specific DNA regions to regulatory proteins and enzymes.

Sequence Complexity in Genomes

Types of DNA Sequences

• Highly Repetitive DNA: Short sequences present in hundreds to thousands of copies, often with no coding function.

• Moderately Repetitive DNA: Includes genes for tRNA, rRNA, and other multi-copy genes (10s–100s of copies).

• Unique DNA: Most protein-coding genes, present in one or a few copies per genome.

Sequence Complexity: Refers to the proportion of unique versus repetitive sequences in a genome. More complex genomes have a higher proportion of unique sequences.