CHAPTER 4 THE TRANSMISSION OF DNA AT CELL DIVISION

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1 1 CHAPTER 4 THE TRANSMISSION OF DNA AT CELL DIVISION Questions to be addressed in this chapter: 1. How is DNA replicated? 2. Following DNA replication, what process ensures that each daughter cell gets a complete copy of the genetic material? 3. During sexual cell division, what process ensures that each gamete receives a representative half of the genetic material? Terminology (see also Glossary pages and web site Semi-conservative DNA replication in which each strand of parental DNA serves as a template for new DNA synthesis Origin the start point of DNA replication Leading strand - the DNA strand that is being synthesized in the same direction as the replication fork is proceeding Lagging strand the DNA strand that is being synthesized in the opposite direction as the replication fork is proceeding Chromatid one of 2 daughter DNA molecules produced by chromosome replication Mitosis cell division that produces 2 daughter cells with identical nuclei to the parent cell Meiosis a process consisting of 2 consecutive cell divisions that produces daughter cells (gametes or sexual spores) with half the genetic material of the parent Meiocyte a diploid cell about to undergo meiosis Homologous pair chromosomes that pair with each other at meiosis and contain the same genes (may have different alleles) Reduction division - division that results in cells with one member of each homologous pair

2 2 DNA = double helix: James Watson and Francis Crick, How is DNA replicated? Structure suggests mechanism 1) hydrogen bonds between backbones 2) H-bonds between specific bases semi-conservative replication (Figure 4-3) - H-bonds break - exposed bases form H-bonds with new bases, providing a template - new molecule = one parent + one new other possibilities? - conservative - dispersive (newly synthesized DNA is a random mixture of old and new) Experimental evidence: Meselson-Stahl experiment (Box 4-1) Hypothesis: if DNA replication is semi-conservative, a label present on both halves of the parental DNA helix should be present in one-half of the daughter DNA helix Experiment: - grew E.coli for many generations in medium containing a heavy isotope of N (N 15 ), so that DNA contained only N 15 - moved to medium containing normal N (N 14 ) and allow DNA to replicate once - centrifuge newly replicated DNA in CsCl gradient - predicted results if replication is: a) semi-conservative or b) conservative results: conclusion: Semi-Conservative Replication must be: -efficient, few mistakes, linked to cell cycle

3 3 DNA polymerization (see figures 4-4, 4-5, 4-6, 4-7) Step 1 Opening the helix > replication fork - origin - in prokaryotes, a single origin/chromosome, in eukaryotes, multiple origins/chromosome - the protein DnaA - recognizes origin and opens - single stranded binding proteins - keep helix open Step 2 a) priming DNA synthesis (Figures 4-6, 4-7) - what does first nucleotide attach to? RNA primer provides the 3 OH - enzyme = primase, major component of the set of proteins that make up the primosome b) synthesizing new DNA (Figures 4-4, 4-5, 4-7) - DNA polymerase III - adds new bases to 3 OH - acts 5 ---> 3 - bases complementary to template Leading and Lagging Strands (Figures 4-5, 4-6, 4-7) - Replication proceeds bidirectionally from the origin, to produce 2 replication forks At each replication fork, 2 strands are being synthesized: - leading strand = - lagging strand = - new primers - Okasaki fragments - DNA polymerase I -removes primer and replaces it with DNA

4 4 - DNA ligase - makes bond between Okasaki fragment and DNA replacing primer Torsion generated: as helicase separated the two strands of DNA at the replication fork, torsion (=supercoiling) is generated ahead of the replication fork This must be removed by the action of gyrase (topoisomerase), that cuts the DNA strands, allows them to unwind, and then reanneals the DNA Mistakes? 1. subunit of DNA polymerase III = ε subunit 2. recognizes mispairs 3. exonuclease + polymerase Ending replication in circular and linear molecules: 1. Rolling circle replication (Plasmids, Figure 4-10) 2. telomeres (linear chromosomes, Figures 4-11, 4-12, 4-13) End result - two identical DNA molecules, each with one parental strand (template) and one newly synthesized strand - in eukaryotes, each daughter is a sister chromatid 2. Following DNA replication, what process ensures that each daughter cell gets a complete copy of the genetic material? - in prokaryotes, reproduction is through cell division (Figures 4-14, 4-15) - our discussion will focus on the eukaryotic mechanism: The cell cycle Four stages:

5 5 S = DNA Synthesis G1 = Gap 1 G2 = Gap 2 M = Mitosis before DNA replication (G1 and S phase see figures 4-14 and 4-24) - chromosomes are single-stranded = 1 duplex DNA molecule = one chromatid centromere single chromatid = 1 DNA molecule after replication (G2 phase) - each chromosome is double stranded, consisting of 2 DNA molecules (chromatids) linked by a condensed region, the centromere Asexual cell division = mitosis in eukaryotes asexual cell division involves no cell fusion

6 6 - after cell division, each chromosome is single stranded, consisting of one duplex DNA molecule or chromatid Question: how does cell division separate chromatids non-randomly, such that each daughter gets a copy of each chromosome? Solution: 1) hold chromatids together while new cells are forming centromere -holds chromatids together until it separates during mitosis 2) separate chromatids in an organized fashion spindle fibres = microtubules - attach to centromeres via kinetochore and move chromatids apart (Figures 4-18, 4-19) With respect to mitosis, the cell cycle may be divided into 2 Stages: 1) interphase (G1, S, G2) replication of DNA, growth of cell 2) Mitosis (M) (Figures 4-20, 4-21) 1) prophase - condensation of chromosomes - breakdown of nuclear membrane 2) metaphase - formation of spindle (microtubules) - attachment of 2 spindles to each centromeres - movement of chromosomes to middle of cell = metaphase plate 3) anaphase - division of centromeres - separation of chromatids 4) telophase - reformation of nuclear membrane

7 7 Sexual Reproduction (Figure 4-16) - involves cell fusion Consider human cycle: IF gametes were produced through standard cell division (mitosis), they would have the same number of chromosomes as cells in the parents. Fusion of gametes would lead to offspring with double the number of chromosomes as the parents. Question: How is the chromosome number maintained in offspring relative to parents? Solution: gamete must have 1/2 the number of chromosomes that the parent does 3. During sexual cell division, what process ensures that each gamete receives a representative half of the genetic material? - cell division that gives rise to gametes must half the number of chromosomes = meiosis (sexual cell division - Figure 4-20) homologous pair - 2 chromosomes which have the same array of genes, although they may have different alleles of those genes diploid individuals: contain a pair of each chromosome (2n) haploid individuals: contain only one member of each homologous pair (n) Haploid and Diploid life cycles -both involve meiosis to form haploid cells, and fusion of those cells to generate a diploid organism -difference is whether mitosis occurs in the haploid (haploid life cycle) or diploid (diploid life cycle) stage of the life cycle. MITOSIS

8 8 - produces 2 identical cells - each cell must contain 1 copy of each chromatid - chromatids (copies) held together by centromere during alignment - chromatids separated to poles by spindle fibres MEIOSIS - produces 4 haploid cells from 1 diploid cell - each product must receive one member of the homologous chromosome pair - At completion of meiosis, 4 haploid gametes are produced, each containing a singlestranded chromosome (1 chromatid) of one member of each homologous pair (for comparison of mitosis and meiosis, see Figures 4-20, 4-24) Question: How does meiosis ensure that each daughter cell receives one member of each homologous pair? Solution: Have 2 divisions, one which separates the homologous chromosomes in an ordered fashion, the second which separates the chromatids in an ordered fashion (Figure 4-20): Division 1 (Meiosis I) = reduction division KEY EVENT: align homologous chromosomes, separate one homologue to each pole Division 2 (Meiosis II) - similar to Mitosis KEY EVENT: align chromosomes (2 chromatids held by centromere), separate one chromatid to each pole Meiosis I Prophase I (Figure 4-22) - homologous chromosomes pair, recombination or crossing over occurs (exchange of chromosome segments) leptotene - chromosomes become visible zygotene - pairing of homologues, crossing-over (Figure 4-23)

9 9 pachytene - synaptonemal complex complete diplotene - slight separation to form chiasmata diakenesis - further contraction Metaphase I - homologous pairs move to equatorial plate - centromeres attach to spindle Anaphase I - one homologue moves to each pole - chromatids remain attached by centromeres, therefore do not separate Telophase I - chromosomes become diffuse - +/- formation of nuclear membrane, cytoplasmic division Meiosis II (similar to Mitosis) Prophase II - chromosomes contracted Metaphase II - chromosomes at equatorial plate, attachment of centromeres Anaphase II - centromeres split, chromatids to opposite poles Telophase II - reformation of nucleus Products of Meiosis I - 2 cells with one member (2 chromatids) of each homologous pair = n Products of Meiosis II - 4 cells, with one member (1 chromatid) of each homologous pair = n (For summary and comparison of Mitosis and Meiosis, see Figures 4-20, 4-24)

10 10 Life cycles (Figure 4-16) haploid life cycle predominant stage is haploid diploid life cycle predominant stage is diploid - in both life cycles, a diploid meiocyte produces four haploid daughter cells - both life cycles alternate between diploid and haploid stages, and employ mitosis and meiosis - difference is the stage of the life cycle at which mitosis occurs to create a nontransient organism - in haploid life cycle, at haploid stage - in diploid life cycle, at diploid stage - phenotype and genotype are normally scored in the non-transient phase of the cycle 4. What determines how alleles of different genes are transmitted from one generation to the next? (Figure 4-20)