Fundamentals of Cell Biology

1. Fundamentals of Cell Biology Chapter 7: The Nucleus and DNA Replication

2. Chapter Summary: The Big Picture (1) • Chapter foci: • Examine the anatomy of the nucleus • Introduction to the protein complexes necessary for building, replicating, and maintaining the structure of DNA • Discussion of mitosis and the importance of the cytoskeleton in regulating mitosis

3. Chapter Summary: The Big Picture (2) • Section topics: • The nucleus contains and protects most of a eukaryotic cell’s DNA • DNA replication is a complex, tightly-regulated process • Mitosis separates replicated chromosomes

4. The nucleus contains and protects most of a eukaryotic cell’s DNA • Key Concepts: • The nucleus is a highly-specialized organelle committed primarily to protecting, copying, and transcribing DNA. • The interior of the nucleus is highly compartmentalized. • DNA copying, plus transcribing and splicing of RNA, are accomplished by large, highly-specialized molecular complexes.

5. The nuclear envelope is a double membrane structure • the nuclear envelope encloses the nucleoplasm • outer membrane of the nuclear envelope is continuous with the ER Figure 07.01: Electron micrograph of the nucleus of a white blood cell (lymphocyte). Figure 07.03: The nuclear envelope is continuous with the endoplasmic reticulum.

6. Nuclear pore complexes regulate molecular traffic into and out of the nucleus • layers of rings stacked on top of one another that span the nuclear membranes, linked to filamentous protein fibrils to form a basket structure • structure undergoes complex conformational changes when it transports material into and out of the nucleus Figure 07.04: The nuclear pore complex appears to be constructed from modular components. Electron micrographs of these components are shown at different stages.

7. The interior of the nucleus is highly organized and contains subcompartments • nucleolus contains DNA that encodes ribosomal RNAs • nucleoli are sites of high transcriptional activity for rRNA genes Figure 07.05: Light micrograph of a human cancer cell with two nucleoi in its nucleus.

8. The nuclear matrix helps to organize chromosomes • chromosomes are compartmentalized into regions called chromosome territories • nuclear matrix helps control the shape of chromosomes and regulate heterochromatin and euchromatin Figure 07.06: Individual chromosomes occupy distinct areas of the nucleus called chromosome territories. Figure 07.07: The nuclear matrix is a network of filaments bound to the nuclear envelope and to DNA.

9. DNA replication occurs at sites called replication factories • DNA replication factories form large complexes in the nucleus devoted to copying DNA with 100% accuracy and no breaks • Replisome is the smallest functional unit in the factories and are responsible for copying one segment of DNA Figure 07.08: DNA replication factories appear as bright spots of fluorescently tagged, newly synthesized DNA.

10. RNA polymerase complexes and spliceosomes are distinct structures within the nucleus • RNA polymerase complexes are responsible for transcribing the DNA sequence in genes into mRNA, rRNA, tRNA, and other RNAs • Spliceosomes are responsible for splicing the newly synthesized RNAs into their mature form

11. DNA replication is a complex, tightly-regulated process • Key Concepts (1): • DNA replication in all organisms is performed by a small number of highly-conserved proteins. • Both prokaryotes and eukaryotes express different forms of DNA polymerase, the enzyme responsible for synthesizing DNA. • DNA replication begins at specific sites called origins of replication. • During replication, double-stranded DNA is unwound and dissociated into single strands that serve as templates for synthesis of complementary DNA strands.

12. DNA replication is a complex, tightly-regulated process • Key Concepts (2): • DNA synthesis occurs only in the 5’-to-3’ direction. • DNA polymerases must bind a double-stranded portion of a DNA molecule to begin synthesis. Most often, the double strand consists of the template DNA strand and a short, complementary RNA primer. • DNA ligase connects individual pieces of newly synthesized DNA to form a complete strand. • The enzyme telomerase adds extra DNA to the ends of chromosomes to protect them from degradation.

13. Cells adhere to one another via specialized proteins and junctional complexes • Key Concepts (4): • Neural cell adhesion molecules (NCAMs) are expressed only in neural cells and function primarily as homotypic cell–cell adhesion and signaling receptors. • Selections are cell–cell adhesion receptors expressed exclusively on cells in the circulatory system. They arrest circulating immune cells in blood vessels so that they can crawl out into the surrounding tissue.

14. DNA polymerases are enzymes that replicate DNA Figure 07.09: The common organization of DNA polymerases is often compared to the human hand.

15. DNA polymerases • DNA polymerases add deoxyribonucleotides to the 3’ end of DNA strand • DNA polymerases proofread their work Figure 07.10: DNA is synthesized in the 5’-to-3’ direction to permit DNA polymerase to proofread the new strand.

16. DNA replication is semi-discontinuous • DNA replication begins at sites on chromosomes called origins of replication • During replication, specialized proteins unwind and separate the two strands to form a replication fork Figure 07.11: A model of the origin recognition complex in yeast, a simple eukaryote. Numerous proteins are contained in the complex. Figure 07.12: A hexameric helicase moves along one strand of DNA.

17. DNA replication is semi-discontinuous • DNA replication requires an RNA primer • leading/lagging strand • Okazaki fragments • DNA ligases join fragments of single-stranded DNA Figure 07.15: Fusion of replication bubbles.

18. Replication of DNA at the end of chromosomes requires additional steps Figure 07.16: Four features of telomerase.

19. Cells have two main DNA repair mechanisms • Excision repair systems • Mismatch repair • Recombination repair Figure 07.17: Excision repair replaces a damaged strand.

20. Mitosis Separates Replicated Chromosomes • Key Concepts: • The function of mitosis is to safely separate replicated chromosomes into two daughter cells. • Mitosis is divided into five phases, based largely on morphological changes in the location and arrangement of chromosomes. • The microtubule cytoskeleton, including microtubule motor proteins, is essential for proper segregation of chromosomes. • The actin cytoskeleton is required for the actual division of one cell into two daughter cells following mitosis.

21. Mitosis is divided into stages • 1879 - Walther Flemming described the motion of what he saw under microscope as “threads” (Greek, mitos) moving in an actively dividing cell Figure 07.18: The events of mitosis.

22. Prophase prepares the cell for division Figure 07.19: The first frame of a video that follows the chromosomes through the initial stages of mitosis.

23. Motors contribute to the formation of the mature spindle in prophase • Dynein motor proteins • Kinesin-related motor proteins Figure 07.20: Microtubule motors help form the mitotic spindle.

24. Chromosomes attach to the mitotic spindle during prometaphase • Kinetochores attach chromosomes to the mitotic spindle Figure 07.23: Once attached to the spindle, a kinetochore can exist in one of two activity states. Figure 07.21: The structure of kinetochores.

25. Arrival of the chromosomes at the spindle equator signals the beginning of metaphase • Metaphase plate = spindle equator • Chromosome recombination takes place during metaphase

26. Separation of chromatids at the metaphase plate occurs during anaphase • The onset of anaphase requires dissolving the connections between sister chromatids • APC • Anaphase is subdivided into two phases: • anaphase A • anaphase B Figure 07.25: As the chromosomes move to the poles (anaphase A), the poles themselves move farther apart (anaphase B), increasing the separation between them.

27. Telephase - Cytokinesis • The structural rearrangements that occur in prophase begin to reverse during telephase • Cytokinesis completes mitosis by partitioning the cytoplasm to form two new daughter cells • Fragmentation of non-nuclear organelles ensures their equal distribution in the daughter cells