Chapter 7: DNA Structure and Replication
Know the assembly of polynucleotide chains
1. Individual nucleotides are assembled into chains by the enzyme DNA polymerase 2. It catalyzes the formation of a phosphodiester bond between the 3′ hydroxyl group of one nucleotide and the 5′ phosphate of an adjacent one 3. Each polynucleotide chain has a sugar-phosphate backbone, consisting of alternating sugar and phosphate groups
What are the 3 properties the genetic code has to have?
1. Must replicate 2. Must encode information 3. Must be able to change (mutable)
What causes the gap in the telomeres and what enzyme fixes this problem? Know basics of telomerase activity steps
GAP - No preceding Okazaki fragment with 3'-OH
Know the essence of Griffith's in vivo transformation experiments. followed by Avery MacLeod-McCarty in vitro transformation experiments MAIN POINTS
Griffith proposed the transformation factor as the molecule that transformed the RII into SIII Avery, MacLeod, and McCarty used heat-killed SIII bacteria and live RII bacteria to infect mice
IIR vs IIIS features (which is virulent or outcome for mice injected with it)
IIR - avirulent, IIIS - virulent
What are Okazaki fragments and how are they combined after being synthesized? Know enzyme involved and what strand they are located on.
Lagging strand forms Okazaki fragments composed of RNA primer and ~1000 bases of DNA. Okazaki fragments are joined together by DNA ligase
What are the similarities and main differences in DNA replication between prokaryotes and eukaryotes?
Similarities: Double-stranded DNA unwound at replication origins Replication forks are formed Bidirectional synthesis creates leading and lagging strands Difference: Eukaryotic DNA replication is more complex There is more DNA than in prokaryotic cells The chromosomes are linear The DNA is complexed with proteins
What are Chargaff's rules and how do they correlate to the base composition studies critical conclusions?
T=A, C=G T+C (pyrimidines)=G+A (purines) The sum of (G+C) does not necessarily equal the sum of (A+T)
The most commonly used DNA polymerase, ______, is isolated from Thermus aquaticus, which occurs naturally in hot springs
Taq
Name the main characteristics of telomeric sequences. How are telomeres important in chromosomes?
The TTAGGG DNA repeat proteins 1 and 2 Cap eukaryotic chromosomes and preserve their integrity & stability Protect chromosomes from end-to-end fusions Protect chromosomes from exonucleases & degradation
What is the difference between complementary and antiparallel? Be able to identify if given sequences. How are they important for DNA functions?
The bases of one strand are complementary to the bases in the corresponding strand (A pairs with T and G pairs with C). The two strands are antiparallel with respect to their 5′ and 3′ ends. DNA replication, transcription, and repair Complementarity important for replication, gene expression, meiotic pairing, molecular hybridization, and more
Be able to determine a consensus sequence
The consensus sequence is identified by determining which nucleotide is used most frequently at each position. For the two nucleotides that occur at an equal frequency at the first position, both are listed at that position in the sequence and identified by a slash mark: T/A G C A A T T.
Be able to identify a replication fork, the 5'-3' orientation of the original DNA strands; show RNA primers, DNA polymerase, Okazaki fragments, and directions of replication on lagging and leading strands.
see image
What is the function of the sliding clamp?
The sliding clamp anchors the DNA pol III core enzyme to the template It is key to the high level of pol III activitycan close around the double-stranded DNA during replication
Explain the Meselson-Stahl experiments and how they ruled out the conservative and dispersive replication models in bacteria. Light and heavy N...
1. E.coli added to heavy(15Nh4Cl) solution let it mix and then added to light 14-N chain medium and let mix/replicate. 2. conservative would have not mixed heavy and light chains (IT DID=disproved) 3. dispersive-would have seen a 3:1 ratio light to heavy chain. DID NOT=DISPROVED Must be semiconservative!
The ______ IIR cells were _______ into _______ IIIS cells This lead to the discovery of?
avirulent, transformed, virulent the transformation factor
Transformation is used by ______ to transfer DNA
bacteria
Define base stacking, major and minor grooves, and identify the 3 forms of DNA and their properties.
base stacking: the offsetting of adjacent base pairs so that their planes are parallel major grooves: 12Å wide and alternates minor grooves: 6Å wide grooves are regions where DNA binding proteins can make direct contact with nucleotidesDNA CONFORMATIONS: B-DNA: Watson-Crick model. Biologically significant conformation aqueous, low-salt conditions; most common and has a right-handed twist of the helix, as does the A-form A-DNA: high-salt or dehydration: bases tilted in relation to the axis, minor/major grooves modified. Mainly in vitro conditions. occasionally detected in cells and is common in bacteriophage Z-DNA: left-handed double helix. Diameter 18 Å, 12 bases per turn, zigzag conformation, no major groove. Robert Wells and colleagues in 1970 and is commonly found near transcription start sites zigzag appearance due to its left-handed twist of the helix
When did transformation not occur and this led to what discovery?
did not occur when treated with DNase meaning that DNA was the transforming factor
Explain the Hershey and Chase bacteriophage experiments and their importance for the discovery that DNA carries the genetic information in prokaryotes. MAIN POINTS
in 1952, Hershey and Chase showed that DNA is responsible for bacteriophage infection of bacteria cells bacteriophages are viruses that infect bacteria
What are the differences between Nucleosides and nucleotides? Be able to identify if given the name of one as an example and if it is a nucleoside or nucleotide. (Hint the ending will help you)
nitrogenous base + pentose sugar = NUCLEOSIDE (end in -ine) Ex. adenosine nucleoside + phosphate group = NUCLEOTIDE (end in -acid) Ex. adenylic acid
DNA contains large amounts of ___________ while proteins contain large amounts of ____________
phosphorus, sulfur
In the 1940's ___________ was believed to be the genetic material
protein
The transforming factor carried hereditary information but could not identify the molecule which lead to....
Avery-MacLeod-McCarty in vitro experiments
Why does proofreading need to occur? What activity allows DNA Polymerase to work?
DNA proofreading to correct occasional errors.Proofreading ability of DNA polymerase enzymes is due to a 3′-to-5′ exonuclease activity
Avery, MacLeod-McCarty used extracted heat-killed SIII bacteria that was divided into aliquots and treated to destroy either....
DNA, RNA, proteins, or lipids and polysaccharides
Differences between dNMPs and dNTPS.
Deoxynucleotide monophosphates (dNMP)-are part of a polynucleotide chain; have single phosphates;N refers to any of the four bases Deoxynucleotide triphosphates (dNTP)-not part of a polynucleotide chain
_______________ are not part of a polynucleotide chain
Deoxynucleotide triphosphates (dNTPs)
Name the three theoretical models of DNA replication. Are they all found in nature?
1. Semiconservative DNA replication: each daughter duplex contains one parental and one daughter strand(1:1 light to heavy). 2. Conservative DNA replication: one daughter duplex contains both parental strands and the other contains both daughter strands(no mixing of light and heavy) 3. Dispersive DNA replication: each daughter duplex contains interspersed parental and daughter segments(3:1 light to heavy chains) only semiconservative DNA found in nature
What are the 3 proofs that DNA is the genetic material in Prokaryotes?
1. Transformation studies between bacteria 2. Transformation studies between bacteria 3. Transfection studies - infecting bacteria directly with phage DNA (purified from phages)
Know the 4 different subunits of DNA Pol III and what function is.
1. core enzyme responsible for polymerization activity. 2 Gamma complex - loads the enzyme onto the template at the replication fork (clamp loader). Requires energy -ATP dependent. 3. β subunit - clamp - prevents the core enzyme falling off the template. 4. τ (tau)- dimerizes two core polymerases to facilitate simultaneous synthesis on both strands.
Lis the 3 main points of the transforming principle
1. transforming agent is DNA 2. transformation is a genetic event 3. broad range of variation
The resulting phage ghosts being unlabeled and the infected bacteria being labeled with _________, proved that the genetic material in T2 phages is ________
32 P, DNA not protein
Phage proteins were labeled with ________, while phage DNA was labeled with _______
35 S, 32 P
Why do prokaryotes only have one origin of replication, while eukaryotes have several of them?
Because eukaryotic chromosomes are linear and much larger than prokaryotic ones, there are multiple origins of replication in the eukaryotic genome during replication. This means that replication can occur simultaneously in hundreds to thousands of locations along each chromosome.
Cancer cells maintain ________ activity and are __________.
Cancer cells maintain telomerase activity and are immortalized.
In the process of bacterial transformation, what is the transforming agent and why is it so?
DNA transformation is a genetic event - changes are transferred into next generations
Do prokaryotes have telomeres?
No because they have circular DNA
Regarding DNA replication in prokaryotes, explain the following terms: a. Origin of replication b. Replication fork c. Circular chromosome d. Replication bubble
Origin of replication -BACTERIA ONLY HAVE 1. DNA replication is most often bidirectional, proceeding in both directions from a single origin of replication in bacterial chromosome Replication fork -At each end of the replication bubble is a replication fork; replication is complete when the replication forks meet Circular chromosome -Bacteria have circular chromosomes Replication bubble-Expansion around the origin of replication, forming a replication bubble, once replication gets under way in bacteria
PCR amplification and dye terminator sequencing similarities and differences.
PCR - Amplification by PCR doubles the number of copies of the targeted DNA sequence each cycle. Uses: A double-stranded DNA template containing the target sequence to be amplified A supply of the four DNA nucleotides A heat-stable DNA polymerase Two different single-stranded DNA primers A buffer solution Dye terminator sequencing: DNA polymerase is used with fluorescent nucleotides to generate new DNA sequences that can be read by a laser and advanced imaging.
Be able to identify the characterization and functions of the 5 types of DNA Polymerase and what properties they have. a. Table 10.2
Polymerase I : more in cell than II or III removes primer & fills gaps Polymerase II: repairs DNA damage caused by external forces repairs at replication fork Polymerase III: the largest 900 kD in vivo 5'- 3' DNA replication proofreading (main thing with DNA polymerization) Polymerase IV/V: Repair of DNA damages caused by external forces (UV, mutagens, radiation, etc.)
Know the steps of DNA replication in their correct order and know the basics for each step.
Prokaryotes: single origin of replication site(OriC)replication is bidirectional-two replication forks form and move in opposite directions until they meet at termination site Eukaryotes: ALL dNTPs must be present. Primer(starting point) ,template strand must all be present, DNA polymerase 1. A dNTP is added to the primer by DNA polymerase 1 during DNA synthesis in the presence of magnesium. and releases inorganic pyrophosphate which drives rxn. direction of synthesis is 5' to 3'.
What is the chemical structure differences between RNA and DNA? (Hint: 2'-C)
RNA has an OH attatched the 2' carbon of the pentose sugar whereas DNA only has H.
What is RNA priming?
RNA priming (PRIMASE) is a universal feature of initiation of DNA replication provides the initial 10-12 bases on the new DNA strand. Primase does not need free 3' end.
What are the basics of DNA replication and what are the three attributes shared by all organisms?
SEMICONSERVATIVE BI-DIRECTIONAL MAIN SYNTHESIS - POLYMERASE III DIRECTION 5' - 3' START AT POINT(S) OF ORIGIN3 Attributes: 1. Each strand of the parental DNA molecule remains intact during replication 2. Each strand of the parental DNA molecule remains intact during replication 3. Completion of replication results in the formation of two identical daughter duplexes composed of one parental and one daughter strand
Know the proteins and genes associated with those we went over in class. a. Main group of enzymes: topoisomerases, Helicases, SSB, primase, DNA pols, DNA ligase, RNA primers, etc.
Topoisomerase relaxes the supercoiling Gyrase (Topoisomerase II) - cut and stabilize DNA Helicases - unwind and stabilize PRIMASE - synthesizes a short (10-12 bases) Primase does not need free 3' end. In E. coli, primase joins DnaA, DnaB, and DnaC at oriC.DNA ligase RNA primer - complementary to the DNA template. DNA ligase joins Okazaki fragments. seals the gap among the resulting DNA segments RNA primers start of new dna
What is the most significant nucleoside phosphate and why?
Triphosphates are most significant 1. Precursor molecules for nucleic acid synthesis in the cell 2. Adenosine- and guanosine triphosphates (ATP, GTP) are essential in cell bioenergetics
What causes and controls supercoiling?
Unwinding of chromosomes during DNA replication will create torsional stress, potentially leading to supercoiling of DNA.• Enzymes called DNA topoisomerases catalyze controlled cleavage and rejoining of DNA to relieve supercoiling
Can eukaryotic organisms contain multiple ori on chromosomes? Be able to identify replication bubbles and forks from the electron micrograph.
Yes, eukaryotic organisms can have multiple origins of replication. fork where it forks, bubbles the whole bubble