Cell Biology Study Notes: The Nucleus and DNA Replication

Key Concepts in Cell Biology

• Nucleus structure

• Nucleus import/export

• DNA Replication

• Origins of replication

• Replication machinery

• DNA polymerase

• Okazaki fragments

The Nucleus

Structure and Function

The nucleus is a membrane-bound organelle found in eukaryotic cells. It houses the cell's chromosomes and is the site of DNA replication and transcription. The nucleus is a defining feature of eukaryotes, distinguishing them from prokaryotes.

• Chromosomes are localized and replicated in the nucleus.

• Transcription of DNA into RNA occurs within the nucleus.

Nuclear Envelope

The nucleus is surrounded by a nuclear envelope, which consists of two lipid bilayer membranes (inner and outer) separated by a perinuclear space.

• The outer membrane is continuous with the endoplasmic reticulum (ER).

• Proteins in the outer membrane bind to actin and intermediate filaments (IFs) of the cytoskeleton.

Nuclear Pores and Nuclear Pore Complex (NPC)

Nuclear pores are specialized channels where the inner and outer nuclear membranes fuse, allowing direct contact between the cytosol and the nucleoplasm.

• Nuclear pores are lined with the nuclear pore complex (NPC), a large protein assembly built from about 30 different proteins called nucleoporins.

• The NPC exhibits octagonal symmetry and contains a central granule called the transporter, which facilitates molecular movement across the envelope.

Molecular Traffic Through Nuclear Pores

Molecules enter and exit the nucleus through nuclear pores, which regulate the passage of proteins, RNAs, and other macromolecules.

• Enzymes and proteins required in the nucleus are imported from the cytoplasm.

• RNAs and ribosomal components are exported to the cytoplasm.

• Small particles, molecules, and ions can also pass through the pores.

Active Transport: Nuclear Localization Signals (NLS)

Large proteins and RNAs require active transport to cross the nuclear envelope. This process is mediated by nuclear localization signals (NLS), which are short amino acid sequences (8–30 residues, often rich in lysine and arginine) that direct proteins to the nucleus.

• Example: The NLS of the SV40 large T antigen is a stretch of seven amino acids near the C-terminus.

Nuclear Import via the Ran/Importin Pathway

The Ran/Importin pathway is a major mechanism for nuclear import:

1. A cytoplasmic protein with an NLS is recognized by importin, which binds the NLS and directs the protein to a nuclear pore.

2. The importin-protein complex is transported into the nucleus via the NPC transporter.

3. Inside the nucleus, importin binds to Ran-GTP, causing release of the cargo protein.

4. The Ran-GTP-importin complex is exported back to the cytoplasm.

5. In the cytoplasm, GTP is hydrolyzed, releasing importin.

Maintaining the Ran-GTP Gradient

The directionality of nuclear transport is maintained by a gradient of Ran-GTP:

• High nuclear Ran-GTP is maintained by a guanine-nucleotide exchange factor (GEF).

• In the cytosol, a GTPase activating protein (GAP) promotes GTP hydrolysis by Ran.

Nuclear Export: Ran-Dependent and Ran-Independent Pathways

RNA export is mediated by adaptor proteins containing nuclear export signals (NES), which are recognized by exportins for transport out of the nucleus.

• High nuclear Ran-GTP promotes release of NLS cargo from importin and binding of NES cargo to exportin.

• Nuclear transport factor 2 (NTF2) shuttles Ran-GDP back into the nucleus.

Nuclear Lamina

The nuclear lamina is a meshwork of intermediate filaments (lamins) lining the inner nuclear membrane, providing structural support.

• Lamins are the only intermediate filaments found in some organisms.

• The nuclear matrix or nucleoskeleton is a network of filaments within the nucleus; its exact role is not fully understood.

The Nucleolus

The nucleolus is the site of ribosome subunit assembly within the nucleus.

• Ribosomal subunits are exported through NPCs.

• Fibrils contain DNA being transcribed into ribosomal RNA (rRNA).

• Granules are rRNA molecules being packaged with proteins.

Clinical Connection: Progeria (HGPS)

Progeria is a disease marked by rapid aging, caused by mutations in the nuclear lamin protein, lamin A. In progeria, lamin A is not processed correctly, leading to a weakened nuclear lamina and associated symptoms such as hypertension and atherosclerosis.

• Lamin A is an intermediate filament protein.

• Defective processing leads to abnormal anchoring and nuclear structure.

DNA Replication, Repair, and Recombination

Overview of DNA Replication

All DNA in the nucleus must be duplicated before cell division. This process ensures accurate distribution of genetic material to daughter cells.

• Mitosis: Nuclear division

• Cytokinesis: Division of the cytoplasm

• Sister chromatids: Duplicated chromosomes attached together

Separation of Sister Chromatids

During mitosis, the mitotic spindle separates sister chromatids, which then become individual chromosomes and are enclosed by new nuclear envelopes.

Cell Cycle Phases

DNA synthesis occurs during the S phase of the cell cycle, which is part of interphase. The cell cycle includes:

• G1 phase: Gap before S phase

• S phase: DNA synthesis

• G2 phase: Gap after S phase

• M phase: Mitosis

Semiconservative DNA Replication

DNA replication is semiconservative: each new DNA molecule consists of one parental strand and one newly synthesized strand.

• Proposed by Watson and Crick

Bidirectional DNA Replication

Replication typically proceeds in both directions from the origin, forming replication forks.

• In bacteria, this is called theta (Θ) replication and occurs in circular DNA molecules.

• In eukaryotes, multiple replicons are formed along linear chromosomes.

Origins of Replication

The origin of replication is a specific DNA sequence where replication begins.

• In E. coli, the origin (oriC) is AT-rich and contains tandem repeats.

• Conserved sequences are called consensus sequences.

Replication Initiation Machinery

Bacterial Initiation

• DnaA binds the 9-mer region of oriC, unwinding DNA at the 13-mer sites.

• SSB (single-stranded binding protein) stabilizes unwound DNA.

• DnaB is a helicase that unwinds DNA during replication.

Eukaryotic Initiation

• Origin Recognition Complex (ORC) binds the replication origin.

• Minichromosome Maintenance (MCM) proteins (including helicases) bind the origin.

• Helicase loaders recruit MCM proteins to the ORC.

• The assembled proteins form the pre-replication complex; replication occurs only after licensing and addition of DNA polymerase.

DNA Polymerases

DNA polymerase catalyzes the elongation of DNA chains by adding nucleotides to the 3' hydroxyl end, synthesizing DNA in the 5' to 3' direction.

• Arthur Kornberg discovered DNA polymerase I (Nobel Prize, 1959).

• Bacterial DNA polymerases: I, II, III, IV, V

• DNA polymerase III is the main replication enzyme in bacteria.

• Eukaryotic DNA polymerases: α, δ, ε (nuclear replication); γ (mitochondrial replication); others for repair.

Discontinuous DNA Synthesis and Okazaki Fragments

DNA synthesis on the lagging strand occurs in short segments called Okazaki fragments, which are joined by DNA ligase to form a continuous strand.

• Okazaki fragments are 1000–2000 nucleotides in bacteria, shorter in eukaryotes.

Proofreading and Error Correction

DNA polymerases possess 3'–5' exonuclease activity for proofreading, removing incorrectly paired nucleotides and reducing error rates to a few per billion base pairs.

• Exonucleases degrade nucleic acids from the ends.

• Endonucleases make internal cuts.

Role of RNA Primers in DNA Replication

DNA polymerases require a pre-existing primer to initiate synthesis. Primase synthesizes short RNA primers using the DNA template.

• RNA polymerases can initiate synthesis without a primer.

• On the leading strand, one primer is needed; on the lagging strand, each Okazaki fragment requires a primer.

• RNA primers are removed and replaced with DNA by DNA polymerase I (in bacteria), and fragments are joined by DNA ligase.

Comparison of DNA Forms: A-form, B-form, Z-form

Form	Helix Type	Major Features	Occurrence
A-form	Right-handed	Shorter, wider helix; 11 bp/turn; deep major groove, shallow minor groove	Dehydrated DNA, RNA-DNA hybrids
B-form	Right-handed	Standard form; 10 bp/turn; wide major groove, narrow minor groove	Most cellular DNA
Z-form	Left-handed	Slender, zigzag backbone; 12 bp/turn; flat major groove, narrow minor groove	DNA with alternating purine-pyrimidine sequences

Key Equations

• DNA synthesis direction: $5' \rightarrow 3'$

• Semiconservative replication: $\text{New DNA} = \text{1 parental strand} + \text{1 new strand}$

Example Application

Defects in nuclear lamina proteins, such as lamin A, can lead to diseases like progeria, demonstrating the importance of nuclear structure in cellular function and human health.