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DNA Replication

Overview of DNA Replication

DNA replication is the process by which a cell duplicates its DNA, ensuring that each daughter cell receives an identical copy. This process is fundamental to cell division and inheritance.

Strand Separation: DNA strands are separated at regions called replication forks.
Origins of Replication: Replication begins at one or more fixed sites known as replication origins.
Semiconservative Replication: Each daughter DNA molecule consists of one parental and one newly synthesized strand.

Eukaryotic Replication of a Linear Chromosome

Eukaryotic chromosomes are linear and contain multiple origins of replication to ensure timely duplication.

Bidirectional Replication: Replication proceeds in both directions from several fixed origins.
Fork Progression: Replication forks advance until they meet another fork traveling in the opposite direction.
Timing: Origins are programmed to initiate replication at fixed times during the S phase of the cell cycle.

DNA Polymerases: Enzymes Catalyzing Chain Elongation

DNA polymerases are essential enzymes that synthesize new DNA strands by adding nucleotides to a primer strand.

Polymerase Reaction: DNA polymerase catalyzes the nucleophilic attack by the 3'-OH group at the primer terminus upon the α-phosphate of the incoming deoxynucleoside triphosphate (dNTP) that is base-paired with the template.

Key Reaction:

Phosphodiester Bond Formation: This reaction forms a new phosphodiester bond and releases pyrophosphate ().
Requirements: DNA polymerase requires a DNA template, an RNA or DNA primer, and dNTPs.
Proofreading: DNA polymerase I has both 3'→5' exonuclease (proofreading) and 5'→3' exonuclease (primer removal) activities.

Discontinuous Synthesis on the Lagging Strand

DNA replication is continuous on the leading strand and discontinuous on the lagging strand, resulting in the formation of Okazaki fragments.

Okazaki Fragments: Short DNA fragments synthesized on the lagging strand.
Primase: Synthesizes short RNA primers to initiate DNA synthesis.
DNA Ligase: Joins Okazaki fragments to form a continuous strand.

Nick Translation and Primer Removal

RNA primers must be removed and replaced with DNA to complete replication.

Nick Translation: DNA polymerase I removes RNA primers and replaces them with deoxynucleotides using its exonuclease and polymerase activities.

Schematic View of the Replication Fork

The replication fork is a complex structure where multiple proteins coordinate DNA synthesis.

Key Components: DNA polymerase, primase, helicase, single-stranded DNA-binding proteins (SSB), topoisomerase, and DNA ligase.
Leading and Lagging Strands: The leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously.

Helicase-Mediated DNA Unwinding

Helicase is responsible for unwinding the double-stranded DNA ahead of the replication fork.

ATP-Dependent: Helicase uses ATP hydrolysis to separate DNA strands.
Torsional Stress: Unwinding causes overwinding (supercoiling) ahead of the fork, relieved by topoisomerase.

Transcription in Eukaryotic Cells

RNA Polymerases and Transcription Factors

Eukaryotic transcription involves three distinct RNA polymerases, each responsible for synthesizing different classes of RNA.

RNA Polymerase I (Pol I): Transcribes major ribosomal RNA (rRNA) genes.
RNA Polymerase II (Pol II): Transcribes protein-coding genes (mRNA) and some small RNA genes.
RNA Polymerase III (Pol III): Transcribes small RNA genes (e.g., tRNA, 5S rRNA).
Transcription Factors: TF I, TF II, and TF III are required for the initiation of transcription by Pol I, II, and III, respectively.

TFIIIA and Zinc Finger Proteins

TFIIIA is a transcription factor containing zinc finger motifs, which are common DNA-binding domains.

Zinc Finger Structure: The α-helices of zinc finger proteins fit within the major grooves of DNA.
Modularity: Zinc finger proteins can be strung together in series to bind specific DNA sequences.

Structures of Eukaryotic Promoters

Promoters are DNA sequences that define where transcription of a gene by RNA polymerase begins.

TATA Box: The eukaryotic counterpart to the bacterial -10 region; essential for transcription initiation.
Enhancer Regions: Additional regulatory sites that may exist several kilobase pairs upstream from the initiation site.

DNA Looping and Transcription Factor Interaction

DNA looping brings activator proteins into contact with transcription factors and the core transcription machinery.

Pol II Interactions: Interacts with several transcription factors, including TATA box-binding protein (TBP) and TFIIA, -B, -E, -F, and -H.
TBP Function: TBP binds to DNA and bends it by 90°, facilitating the assembly of the transcription complex.

Histone Acetylation and Transcriptional Activity

Histone acetylation is a key epigenetic modification that regulates gene expression.

Acetylation: Addition of acetyl groups to lysine residues on histone tails neutralizes their positive charge.
Effect: Weakens ionic interactions between histones and DNA, making chromatin more accessible for transcription.

Termination of Transcription in Eukaryotes

Termination of transcription by RNA polymerase II involves cleavage of the pre-mRNA and addition of a poly(A) tail.

Cleavage: The pre-mRNA carrying the AAUAAA signal is cleaved 11 to 30 residues downstream of this site.
Polyadenylation: A poly(A) tail is added by poly(A) polymerase, which enhances mRNA stability (the longer the tail, the longer the half-life).

Posttranscriptional Processing

5' Capping of Pre-mRNA

Eukaryotic pre-mRNA is modified at the 5' end by the addition of a 7-methylguanosine cap.

Function: Protects mRNA from degradation and is involved in ribosome binding during translation.

Splicing of Pre-mRNA

Splicing removes non-coding sequences (introns) from pre-mRNA and joins coding sequences (exons).

snRNPs: Small nuclear ribonucleoproteins (snRNPs) such as U1 and U2 aid in pre-mRNA loop formation.
Spliceosome: The complex of snRNPs and pre-mRNA that carries out splicing.

Alternative Splicing

Alternative splicing allows a single gene to produce multiple protein isoforms by varying the combination of exons included in the final mRNA.

Example: The α-tropomyosin gene in rats can be spliced in seven different ways to produce different proteins.

Translation: Protein Synthesis

Overview of Translation

Translation is the process by which ribosomes synthesize proteins using mRNA as a template.

Directionality: mRNA is read 5' → 3', and the polypeptide is synthesized from the N- to the C-terminus.
Participants: mRNA, tRNA, and ribosomes are the major components.

Activation of Amino Acids

Amino acids are activated and attached to their corresponding tRNAs by aminoacyl-tRNA synthetases.

Anticodon Loop: Contains a trinucleotide sequence (anticodon) complementary to the codon in mRNA.

The Genetic Code

The genetic code is a set of rules by which information encoded in mRNA is translated into proteins.

Start Codon: AUG encodes methionine and serves as the start codon in eukaryotes.
Stop Codons: UAA, UGA, and UAG are stop (nonsense) codons that signal termination of translation.

Formation of Aminoacyl-tRNA

The first step in protein synthesis is the formation of aminoacyl-tRNA.

Step 1: The amino acid is activated by ATP to form aminoacyl adenylate.
Step 2: The activated amino acid is coupled to the tRNA, and AMP is released.

Ribosome Structure

Ribosomes are large ribonucleoprotein complexes responsible for protein synthesis.

Bacterial Ribosome: 70S, composed of 50S and 30S subunits.
Eukaryotic Ribosome: 80S, composed of 60S and 40S subunits.
Sites: A (aminoacyl), P (peptidyl), and E (exit) sites for tRNA binding and movement.

Mechanism of Translation

Stage 1 – Initiation

Initiation factors (IF1, IF2, IF3) facilitate the dissociation of the 70S ribosome in bacteria.
mRNA and initiator tRNA bind the 30S subunit; the 50S subunit then joins to form the initiation complex.

Stage 2 – Elongation

Peptide chains are attached to the tRNA in the P site; the A site receives the next aminoacyl-tRNA.
Peptide bond formation is catalyzed by peptidyl transferase.
The ribosome translocates, moving the tRNA from the A site to the P site, and the empty tRNA exits via the E site.

Stage 3 – Termination

When a stop codon is encountered, release factors bind to the A site, promoting the release of the polypeptide chain.
The ribosome dissociates from the mRNA, completing translation.

DNA Methylation, Gene Silencing, and Epigenetics

DNA Methylation in Eukaryotes

DNA methylation is an epigenetic modification that plays a critical role in gene regulation, development, and disease.

CpG Islands: Regions of DNA with a high frequency of CpG sites; often found near gene promoters.
Heritability: DNA methylation patterns are heritable and can be maintained through cell division.
Enzymes: Mammalian cells possess three DNA methyltransferases: DNMT1 (maintenance), DNMT3a, and DNMT3b (de novo methylation).
Gene Silencing: Methylation can lead to permanent gene inactivation; demethylation can activate repressed genes.
Biological Roles: DNA methylation is responsible for X chromosome inactivation and genomic imprinting.

Table: Functions of DNA Methylation

Function	Description
X Chromosome Inactivation	One X chromosome in females is inactivated by methylation
Genomic Imprinting	Only one parental allele is expressed; the other is silenced by methylation
Gene Silencing	Methylation of promoter regions prevents gene expression

Example: In cancer, abnormal DNA methylation patterns can lead to inappropriate gene silencing or activation.

Additional info: DNA methylation is a reversible modification and is a major focus of epigenetic research, with implications for development, disease, and therapeutics.