Eukaryotic DNA Replication

• Eukaryotic DNA replication involves multiple large linear chromosomes and origins of replication, typically in AT-rich regions, proceeding bidirectionally with two replication forks per bubble.

DNA Copying Requirements

• Progressive addition of new nucleotides (A, C, T, G) is needed
• A starting point for nucleotide addition
• Unwinding of the double-stranded DNA to create parental templates.
• Release of tension from DNA unwinding
• Prevention of single-stranded DNA from reforming and protection from degradation.
• Joining of newly synthesized fragments on both leading and lagging strands.

Leading and Lagging Strands

• The leading strand is synthesized continuously in the 5’ → 3’ direction.
• The lagging strand is synthesized discontinuously in the 5’ → 3’ direction as Okazaki fragments.
• DNA synthesis occurs towards the replication fork.

Semi-Discontinuous Replication

• Replication is semi-discontinuous due to the different modes of synthesis on the leading and lagging strands.

Primase

• Primase, an RNA polymerase, synthesizes an RNA primer to provide a starting point for DNA polymerization.

DNA Polymerase III

• DNA polymerase III needs a free OH group to attach the phosphate group of an incoming nucleotide.
• It synthesizes DNA exclusively in the 5’→ 3’ direction.
• DNA polymerase III synthesizes new DNA strands complementary to the parental template strands.
• It cannot initiate copying from single-stranded DNA.

Single Stranded Binding Proteins (SSBP)

• SSBPs prevent single strands from reannealing or degrading; DNA polymerase III displaces them during synthesis.

RNase H and DNA Polymerase Activity

• RNase H is an endonuclease that degrades the RNA portion of DNA:RNA hybrids.
• DNA polymerase synthesizes DNA by adding nucleotides complementary to the lagging strand's parental DNA template.

DNA Ligase

• DNA ligase joins Okazaki fragments after RNA primers are removed and replaced with DNA.
• It also joins newly synthesized fragments within replication bubbles, including leading strands.

DNA Error Repair During Replication

• DNA replication has high accuracy due to DNA polymerase III's proofreading.
• The error rate of DNA polymerase III is 1 in 10^8 - 10^10 base pairs replicated.
• DNA polymerase III checks newly inserted bases against the template.

3’ to 5’ Exonuclease Activity

• Incorrect bases are removed by the 3’ to 5’ exonuclease activity of DNA polymerase III

DNA Error Repair After Replication

• Errors in DNA can occur from incorrectly inserted bases as well as radiation/chemical damage
• An endonuclease enzyme removes incorrect or damaged nucleotide bases after replication.

Clonal Reproduction

• Single-celled organisms reproduce by binary fission resulting in genetically identical offspring.

Sexual Reproduction and Meiosis

• The sexual cycle is present in almost all eukaryotes.
• Gametes (sperm and egg) are normally haploid (23 chromosomes).
• Meiosis is a cell division process that halves the number of chromosomes going into gametes.

Meiosis Overview

• Meiosis involves two rounds of cell division but only one round of DNA replication.
• It results in four haploid cells.
• These four haploid cells are genetically distinct.
• Meiosis converts diploid cells into haploid cells.

Meiosis I: Prophase I

• The nuclear envelope breaks down, chromosomes condense, and the spindle forms
• Homologous chromosomes synapse (line up)
• Crossing over occurs between non-sister chromatids at the chiasmata.
• Crossing over is less likely to occur near the centromere.
• Each chromatid is now a mix of DNA from each homologous chromosome.

Meiosis I: Metaphase I

• Chromosomes attach to kinetochore microtubules at each centromere.
• Each pair lines up independently along the metaphase plate.
• Paired homologous chromosomes move to the metaphase plate.
• Chiasmata line up on the metaphase plate.

Meiosis I: Anaphase I

• Recombined homologous chromosomes separate (disjoin).
• Sister chromatids remain attached to each other.
• The cell elongates.
• Each duplicated chromosome moves to opposite ends of the cell.

Meiosis I: Telophase I

• Duplicated chromosomes reach opposite poles.
• The spindle disappears and the nuclear envelope reforms.
• Cells are now haploid

Meiosis I: Cytokinesis

• Cytokinesis involves the formation of a cleavage furrow in animal cells.
• Cytoplasm divides, resulting in two haploid cells.
• Cells (and sister chromatids) are genetically different due to crossing over.

Meiosis II: Prophase II

• There are no chiasmata in meiosis II (no crossing over).
• The spindle forms as centrosomes duplicate and move to opposite poles.
• Kinetochore microtubules attach to each duplicated chromosome at the centromere.
• Each duplicated chromosome is still composed of two chromatids attached at centromeres

Meiosis II: Metaphase II

• Duplicated chromosomes align at the metaphase plate.
• Centromeres also lie on the metaphase plate.

Meiosis II: Anaphase II

• Sister chromatids disjoin at the centromeres, and each chromatid becomes an independent daughter chromosome.
• Daughter chromosomes move toward opposite poles as kinetochore microtubules shorten.
• The nonkinetochore microtubules lengthen, and the cell elongates.

Meiosis II: Telophase II

• Two daughter nuclei (with a nuclear envelope) form in the cell.
• The meiotic division of one parent cell produces four daughter cells, each with a haploid set of unduplicated chromosomes.
• The four daughter cells are genetically distinct from each other and from the parent cell.

Sexual Reproduction and Genetic Diversity

• Sexual reproduction produces genetic diversity through independent assortment of chromosomes, crossing over, and random fertilization of gametes.

Genetic Diversity

• Genetic diversity allows selective responses to spatially variable environments, changing environments, and sib-sib competition.

Karyotypes

• Karyotypes are ordered, visual representations of the chromosomes in a cell, captured during metaphase.
• Chromosomes are cut out of an image of the cell and arranged to create the karyotype display.
• All chromosomes except X and Y are present in homologous pairs.
• Chromosomes are ordered by size, with the exception of chromosome 1.

Homologous Chromosomes, Locus, Gene, and Allele

• Homologous chromosomes are a pair with the same genes, one inherited from each parent.
• A locus is the location of a gene on a chromosome.
• A gene is a DNA region (sequence) that produces a functional RNA molecule and is a unit of hereditary information.
• An allele is an alternative version of a gene.

Cell Cycle

• The cell cycle alternates between the mitotic phase and interphase.

Mitotic Phase

• Distribution of chromosomes into two daughter nuclei.
• Cytokinesis divides the cytoplasm to produce two daughter cells.

Interphase

• Interphase includes G1, S, and G2 phases.
• A duplicated chromosome comprises two genetically identical sister chromatids that separate during mitosis.
• A duplicated G2 chromosome consists of two chromatids, each a double-stranded DNA molecule.

G2 Interphase

• The nuclear envelope is intact.
• The nucleolus is visible, containing one or more nucleoli.
• Two centrosomes form.

Mitosis

• Mitosis produces two genetically identical daughter cells.
• It consists of five phases: prophase, prometaphase, metaphase, anaphase, and telophase.

Mitosis: Prophase

• Nucleoli disappear.
• Duplicated chromosomes condense and appear as two identical sister chromatids joined at their centromeres.
• The mitotic spindle begins to form.
• Microtubules lengthen, and the centrosomes move to opposite poles.

Mitosis: Prometaphase

• The nuclear envelope breaks down, and chromosomes fully condense.
• A kinetochore (protein structure) forms at the centromere of each chromatid.
• Microtubules that attach to the kinetochores are called kinetochore microtubules.
• Nonkinetochore microtubules lengthen the cell by interacting with those from the opposite pole of the spindle.

Mitosis: Metaphase

• Centrosomes are now at opposite poles of the cell.
• Kinetochore microtubules are attached to the kinetochores of all sister chromatids.
• Duplicated chromosomes align at the metaphase plate; homologous pairs do not interact.
• Centromeres lie on the metaphase plate, an equal distance between the spindle’s two poles.
• Karyotypes are made using images of cells in metaphase.

Mitosis: Anaphase

• Sister chromatids disjoin at the centromeres; each chromatid becomes an independent daughter chromosome.
• Daughter chromosomes move toward opposite poles as their kinetochore microtubules shorten.
• Nonkinetochore microtubules lengthen, and the cell elongates.
• Anaphase ends when the two poles of the cell contain identical and complete collections of chromosomes.

Mitosis: Telophase and Cytokinesis

• Chromosomes become less condensed.
• Spindle microtubules break down.
• Two daughter nuclei (with nuclear envelope) form in the cell; nucleoli reappear.
• Mitosis, the division of one nucleus into two genetically identical nuclei, is now complete.
• The cytoplasm divides, resulting in two daughter cells.
• Cytokinesis involves forming a cleavage furrow in animal cells and a cell plate in plant cells.