General Biology 2 BZE: DNA Replication Class Notes PDF

General Biology 2 BZE: Copy that! Class 14: DNA Replication  Today’s programme: 1) DNA replication is semi-conservative Overview Discovery of the model of replication 2) Synthesizing a new strand Overview Replication factory DNA replication complex 3) Proka...

General Biology 2 BZE: Copy that! Class 14: DNA Replication  Today’s programme: 1) DNA replication is semi-conservative Overview Discovery of the model of replication 2) Synthesizing a new strand Overview Replication factory DNA replication complex 3) Prokaryotes vs Eukaryotes Overview Eukaryotes Campbell: Chapter 16 REVIEW: WHY DO CELLS REPLICATE DNA? Faithful reproduction of genetic material prior to mitotic and meiotic cell division for the purpose of: Growth and development Tissue Renewal (ex: skin cells, red blood cells) replication occurs Germ Cell Production during S phase Why do cells replicate? INTERPHASE To make more cells. Fastest amount of cell division occured during embryotic development G1 S G2  1) REVIEW: DNA REPLICATION IS SEMI-CONSERVATIVE 1. Bacteria were cultured in a medium with a heavy isotope of nitrogen. They used it to synthesize DNA. 2. The bacteria were transferred to a new medium with a lighter isotope of nitrogen. 3. DNA was centrifuged after 1 DNA replication was completed by the bacteria. A sample of bacteria was collected. DNA was extracted from each bacterial sample and separated it by density. 4. DNA was centrifuged again nitrogen used as uses bacteria since life after a second replication. cycle 20 minutes, can nitrogenous bases Another sample of easily manipulate bacteria was collected. binary fission DNA REPLICATION IS SEMI-CONSERVATIVE Which model (on the right) best explains the observed data?  2) SYNTHESIZING A NEW STRAND It takes one of your somatic cells just a few hours to copy all of its DNA during S phase of interphase: 46 chromosomes one long double-helical molecule per chromosome ~6 billion nucleotide pairs This replication of an enormous amount of genetic information is achieved with very few errors: Only about one per 10 billion nucleotides. DNA replication is remarkable in its speed and accuracy. SYNTHESIZING A NEW STRAND: PROKARYOTES In optimal conditions, E. coli can replicate their DNA (a single chromosome of ~4.6 million nucleotide pairs) and divide in < 1 hour. The replication of chromosomal DNA begins at particular sites called origins of replication, short stretches of DNA that have a specific sequence of nucleotides. Proteins that initiate DNA replication recognize this sequence and attach to the DNA, separating the two strands and opening up a replication “bubble”. The parent DNA will be copied in both directions from the origin at the same time until the whole chromosome is replicated.  PROKARYOTES: REPLICATION FORK At each end of a replication bubble is a replication fork, a Y-shaped region where the parental strands of DNA are being unwound. Since replication will happen in replication forks both directions from the origin, there are 2 replication forks. More than 12 enzymes and other proteins participate in DNA replication. They are clustered together into a “factory” through which DNA passes.  PROKARYOTES: REPLICATION FACTORY The first step of DNA replication is to unzip the double-stranded DNA and keep it apart. Components involved: Helicase (enzyme) ▪ Unzips the pay attention to complementary direction (5 to 3) polynucleotide strands at the origin of replication SSBP (single-strand binding protein) ▪ Adhere to the separated polynucleotide strands to prevent reannealing ▪ Reannealing = strands “zipping” back  PROKARYOTES: REPLICATION FACTORY The second step is to release the tension. Separation of the polynucleotide strands leads to supercoiling in the DNA molecule ahead of the replication fork. Components involved: Topoisomerase (enzyme) ▪ Helps relieve this strain by breaking, swiveling, and rejoining DNA strands.  PROKARYOTES: REPLICATION FACTORY The third step is to start synthesizing the new strand. Components involved: Primase (enzyme) ▪ Synthesizes a primer (short chain of RNA) ▪ Uses the parental DNA as a template. DNA polymerase (enzyme) ▪ catalyzes the polymerization of DNA nucleotides PROKARYOTIC REPLICATION FACTORY: STEP 3  PROKARYOTIC REPLICATION FACTORY: STEP 3 question: where would u add a nucleotide to? The enzyme which catalyzes the polymerization of DNA nucleotides (DNA polymerase) cannot initiate synthesis of a polynucleotide strand. DNA Polymerase can only add nucleotides to the 3’ end of an existing polynucleotide strand.  PROKARYOTIC REPLICATION FACTORY: STEP 4 The fourth step is to synthesize the new complementary DNA strand. Components involved: DNA polymerase III (enzyme) ▪ Adds complementary DNA nucleotides to the free 3’ end of the strand. ▪ Complementary DNA strands can only be elongated in the 5’ → 3’ direction. Sliding clamp ▪ Ensures DNA pol III maintains contact with the template strand The formation of phosphodiester bonds during the polymerization requires energy which is provided by the process. dephosphorylation of nucleoside triphosphates.  PROKARYOTIC REPLICATION FACTORY: STEP 4 Because DNA pol III can only add nucleotides to an open 3’ end, we must investigate the implications on the two replication forks: Leading strand Lagging strand Each replication fork has a leading strand and a lagging strand. Along one template strand, DNA Pol III can synthesize a complementary strand continuously in the mandatory 5’ → 3’ direction. This new DNA strand is called the leading strand. To elongate the other new strand in the mandatory 5’ to 3’ direction, DNA pol III must work along the other template strand in the direction away from the replication fork. The new DNA strand synthesized in this way is called the lagging strand.  STEP 4: LEADING STRAND Only one RNA primer is required for each leading strand it will be synthesized continuously in same the direction of the replication fork’s movement. As the parent DNA unwinds, the opening space in front of the leading strand allows continuous adding of nucleotides to an open 3’ end.  STEP 4: LAGGING STRAND the lagging strand is synthesized discontinuously, as a series of segments. Lagging strands grow in the last one opposite direction from the movement of the replication fork. They are produced in short will be a diagram and have to determine which lagging strand was first made segments called Okazaki Fragments as the parent template strand is slowly more and more exposed. Each Okazaki fragment requires a new primer.  STEP 4: LAGGING STRAND DNA Polymerases can only add nucleotides to the 3’ end of a polynucleotide strand which has and exposed hydroxyl group. So even though the replication fork is moving away from the origin, the lagging strand can only grow by Okazaki fragments in the direction towards the origin of replication.  REPLICATION FACTORY: STEP 5 The fifth step is to remove the RNA primers and replace them with DNA nucleotides. Components involved: Rnase H (enzyme) ▪ Recognizes RNA-DNA hybrid segments ▪ Degrades the RNA primer by hydrolyzing its phosphodiester bonds. DNA polymerase I RNase H removes primer and (enzyme) DNA pol I adds DNA ▪ Adds DNA nucleotides nucleotides. to any exposed 3’ ends on the leading and lagging strands.  REPLICATION FACTORY: STEP 6 The sixth step is to glue the fragments of lagging strands together. Components involved: DNA ligase (enzyme) ▪ Attaches adjacent fragments of DNA on the newly synthesized strands to make a continuous polynucleotide strand. RNase H removes primer and DNA pol I adds DNA nucleotides. REPLICATION FACTORY: STEP 6 Primase Rnase H DNA polymerase I DNA ligase REPLICATION FACTORY: SUMMARY RNase H removes primer and DNA pol I adds DNA nucleotides.  DNA REPLICATION COMPLEX The various proteins that participate in DNA replication actually form a single large complex, a “DNA replication machine.” Many protein-protein interactions facilitate the efficiency of this complex. The DNA replication complex may not move along the DNA; rather, the DNA may move through the complex during the replication process. coordinated, leading won’t be ahead 3) PROKARYOTES vs EUKARYOTES bacteria circular, only one origin of replication, one replication bubble  DNA REPLICATION: EUKARYOTES Eukaryotic DNA contains many more base pairs: (6 billion in humans) compared to prokaryotic DNA (~ 5 million). Replication therefore takes longer than for prokaryotes (a few hours in humans). Eukaryotic DNA is in multiple linear strands unlike the circular DNA of prokaryotes. The ends of these linear strands are vulnerable to damage and are therefore protected by telomeres (DNA extensions). won’t be made into proteins  EUKARYOTES: TELOMERES Telomeres are short repeated DNA sequences (TTAGGG) at both ends of a linear DNA strand which contain no genes. In humans, chromosomes have between 100 and 1000 TTAGGG sequences at each end. Telomeres get shorter with each replication event! They serve two protective functions: Specific associated proteins prevent the activation of DNA damage signaling. Provides some protection against the organism’s genes shortening.  EUKARYOTES: TELOMERES Telomeres get shorter which each replication event The end of the molecule on each lagging strand is left un-replicated and the exposed section on the parent strand breaks off. Telomeres acts as a buffer. However, telomeres do not completely prevent the erosion of genes near the ends of chromosomes; they merely postpone it.  EUKARYOTES: TELOMERES Some evidence showing that shortening of telomeres is correlated to the aging process of certain tissues and even to aging of the organism. The idea of a “telomere clock” acting as a cellular timekeeper of aging was first proposed by Canadian researcher Calvin Harley in 1990. Harley and his colleagues showed that some premature aging syndromes are associated with decreased telomere length. What is happening in germ cells?  EUKARYOTES: TELOMERASE ENZYME Telomerase is an enzyme which can extend the length of telomeres. It is only found in: Germ cells (undergo meiosis to make gametes) Cells within embryos/fetuses Certain tumor cells Stem cells in adults don’t have telomerase, so the ends of their DNA get shorter with time. Normal shortening of telomeres may protect organisms from cancer by limiting the number of divisions that somatic cells can undergo. Many cancer cells seem capable of unlimited cell division, as do immortal strains of cultured cells.  A Cure for Aging? Mice overexpressing telomerase are healthier longer if cancer suppressing genes are overexpressed as well EMBO Mol Med. 2012 August; 4(8): 685–687  DNA REPLICATION: PROKARYOTES vs EUKARYOTES Characteristics Prokaryotes Eukaryotes Circular form Chromosomes Linear form (some exceptions) Origin of replication One per chromosome Many per chromosomes DNA polymerase III Over 11 different DNA polymerases DNA polymerase I polymerases discovered Okazaki fragments Present (longer) Present (shorter) Telomeres Absent (not needed) Present (necessary) Now, you should be able to: Describe each step of DNA replication, the enzymes involved and their functions Explain the main differences between prokaryotic and eukaryotic DNA replication Explain the function of telomerase and the purpose of telomeres in eukaryotic DNA