DNA Replication PDF

DNA Replication Recorded Lecture, Part 1 Today’s LOs Identify the 5’ and 3’ ends of a nucleotide on a DNA strand State which end (5' or 3') of a DNA strand that new nucleotides are always added to Explain how the definition of antiparallel is reflected in DNA structure Given tha...

DNA Replication Recorded Lecture, Part 1 Today’s LOs Identify the 5’ and 3’ ends of a nucleotide on a DNA strand State which end (5' or 3') of a DNA strand that new nucleotides are always added to Explain how the definition of antiparallel is reflected in DNA structure Given that DNA is a double-stranded helix, state how many strands of DNA are in a single DNA molecule Describe the process of semiconservative DNA replication Define the origin of replication, the replication bubble, and the replication fork See Blackboard for a complete list of LOs State the functions of key proteins involved in DNA replication, including key enzymes and binding proteins Predict the consequence if a key protein involved in DNA replication were mutated or could not function Explain the process of DNA replication in eukaryotes Differentiate between leading strand and lagging strand Explain why DNA polymerase and primase must lay nucleotides in the 5’→3' direction Explain why DNA polymerase needs a primer, but primase does not Describe the process of DNA replication in bacteria (i.e. circular vs linear DNA replication) Relate the role of telomeres to the shortening of linear chromosomes during each round of replication All life relies on DNA Bacterial DNA Viral DNA  Circular Double-stranded Eukaryotic DNA Linear Double-stranded Linear or circular Single or double-stranded Video Question How many strands of DNA are in the chromosome pictured at right? A. 1 B. 2 C. 4 Remember: DNA is a double-stranded molecule! The two strands are wound together, to make a helix. Before replication, each chromosome contains 1 double-stranded DNA molecule = 2 strands each After replication, each chromosome contains 2 double-stranded DNA molecules = 4 strands each But wait… Video Question What other organelle in all eukaryotic cells contains DNA, and what is that DNA’s structure? A. Golgi apparatus, linear B. Mitochondria, linear C. Chloroplast, circular D. Mitochondria, circular E. Chloroplast, linear Not in all eukaryotes But wait… Eukaryotic DNA here Linear double-stranded Bacterial DNA here Circular double-stranded Endosymbiotic theory mitochondria DNA Structure Double-stranded Complementary Antiparallel 5C 5C 3C 3C 5 end 3 end 5 end 3 end 5C 5C 3C 3C Video Question Given this DNA sequence, what is the nucleotide sequence of the complementary strand? A. -ACTGTTAGATT-5′ B. -GGGACCCGAGA-5′ C. -GGGACCCGAGA-3′ D. -CCCTGGGCTCT-5′ E. -CCCTGGGCTCT-3′ 5’-CCCTGGGCTCT-3’ Remember that it MUST be antiparallel DNA Structure Double-stranded Complementary Antiparallel 5C 5C 3C 3C 5 end 3 end 5 end 3 end 5C 5C 3C 3C What does that mean for DNA replication? OH DNA Synthesis Phosphodiester bond OH Nucleotide 1 Through dehydration reaction OH Nucleotide 2 H H2O How do we replicate a DNA molecule using DNA synthesis? DNA grows at the 3’ end of every strand OH on 3’ C of a nucleotide bonds to a phosphate group on 5’ C on another nucleotide 3’ 5’ Video Question In the diagram of DNA synthesis shown here, the next nucleotide added to the new DNA strand will have a(n) ____ base. A. Adenine B. Guanine C. Cytosine D. Thymine Can only add nucleotides to 3’ end! DNA Replication 3’ 5’ What two things did you have to consider to answer this question? 1. → 3’ synthesis 2. Complementary base pairing New strand on left here A new DNA molecule is synthesized 5’ → 3’ when activated nucleotides (dNTPs) are added to the 3’ end of the strand. Image: bioninja.com DNA drawings vary in how much detail they show Image: bioninja.com This simplified drawing shows how each DNA strand with the original “parent” molecule acts as a template for a new, complementary strand. Image: www.news-medical.net Each new DNA molecule is composed of ½ original template strand (in blue below) and ½ newly synthesized strand (in cream below) We’ll come back to this in Live Class Today’s LOs Identify the 5’ and 3’ ends of a nucleotide on a DNA strand State which end (5' or 3') of a DNA strand that new nucleotides are always added to Explain how the definition of antiparallel is reflected in DNA structure Given that DNA is a double-stranded helix, state how many strands of DNA are in a single DNA molecule Describe the process of semiconservative DNA replication Define the origin of replication, the replication bubble, and the replication fork See Blackboard for a complete list of LOs State the functions of key proteins involved in DNA replication, including key enzymes and binding proteins Predict the consequence if a key protein involved in DNA replication were mutated or could not function Explain the process of DNA replication in eukaryotes Differentiate between leading strand and lagging strand Explain why DNA polymerase and primase must lay nucleotides in the 5’ → 3' direction Explain why DNA polymerase needs a primer, but primase does not Describe the process of DNA replication in bacteria (i.e. circular vs linear DNA replication) Relate the role of telomeres to the shortening of linear chromosomes during each round of replication DNA Replication Recorded Lecture, Part 2 Today’s LOs Identify the 5’ and 3’ ends of a nucleotide on a DNA strand State which end (5' or 3') of a DNA strand that new nucleotides are always added to Explain how the definition of antiparallel is reflected in DNA structure Given that DNA is a double-stranded helix, state how many strands of DNA are in a single DNA molecule Describe the process of semiconservative DNA replication Define the origin of replication, the replication bubble, and the replication fork See Blackboard for a complete list of LOs State the functions of key proteins involved in DNA replication, including key enzymes and binding proteins Predict the consequence if a key protein involved in DNA replication were mutated or could not function Explain the process of DNA replication in eukaryotes Differentiate between leading strand and lagging strand Explain why DNA polymerase and primase must lay nucleotides in the 5’ →3' direction Explain why DNA polymerase needs a primer, but primase does not Describe the process of DNA replication in bacteria (i.e. circular vs linear DNA replication) Relate the role of telomeres to the shortening of linear chromosomes during each round of replication DNA Replication 3’ 5’ Let’s explore how the DNA replication machinery coordinates DNA synthesis DNA Replication Helicase Short DNA sequence = Origins of replication 1. Open DNA molecule Replication bubble DNA Replication Short sequence = Origins of replication Origin of replication Helicase Open DNA molecule → Hold open Helicase Hold open Single-stranded binding proteins Supercoiling On youtube: https://www.youtube.com/watch?v=EYGrElVyHnU Replication bubble DNA Replication Short sequence = Origins of replication Origin of replication Helicase Open DNA molecule → Hold open Helicase Hold open Single-stranded binding proteins Topoisomerase Topoisomerase Topoisomerase releases the tension Topoisomerase creates temporary breaks in DNA ahead of the replication fork and then re-connects DNA This will decrease the number of coils and release the tension in the DNA Middle coil is eliminated Replication fork Helicase Helicase RNA primer Origin of replication Open DNA molecule Hold open →Build new DNA strand (Add RNA primer) Primase DNA Replication Video Question For this DNA sequence to the right, which strand below could act as a primer? A. -ACUGUUAGA-5′ B. -AAAUUUGGC-5′ C. -AUGCUUUGA-5′ D. -GGGACCCGA-3′ E. -CCCUGGGCU-3′ 5. -CCCTGGGCTCTGTAAATGTTTCTAAGTG-3’ 6. -GGGACCCGAGACATTTACAAAGATTCAC-5’ 5′-CCCUGGGCU-3′ The RNA primer must be complementary, and antiparallel to the DNA strand DNA Replication 1. Label the 5’ and 3’ ends on all 4 primers (they’re complementary to the DNA template strand) 2. Draw where the new DNA strand forms and label its 5’ & 3’ ends 3. Draw arrows to point out the overall direction the new strands form from both open DNA strands RNA primer In your notes… DNA Replication RNA primer B A Video Question Where is the 5’ end on this RNA primer? A. A B. B Since this is the 3’ end… Antiparallel rules state that this must be the 5’ end Replication fork DNA Replication Helicase Origin of replication Open DNA molecule Hold open →Build new DNA strand (Add RNA primer) RNA primer Helicase DNA pol III Replication fork DNA Replication Helicase Helicase RNA primer New strand New strand Origin of replication DNA pol III Open DNA molecule Hold open →Build new DNA strand (Add RNA primer) Replication fork DNA Replication Helicase Helicase RNA primer Origin of replication DNA pol III So, what really happens at this portion of the replication bubble? However… RNA primers are always added close to origin of replication, first Primases don’t “wait” for fork to move far away from origin before adding primers, as shown here DNA Replication primer 3’ 3’ Helicase Helicase Origin of replication 5’ 3’ 3’ 1. Add primers (where needed) 2. Continue to build the new DNA molecule where appropriate What happens next? In your notes… DNA Replication 3’ 3’ Helicase Helicase Origin of replication 5’ 3’ 3’ A B C D Video Question To continue building a new molecule, which locations need more primers to continue replication than the ones already shown below? A. & D B. & B C. & C D. & D DNA Replication 3’ 3’ Helicase Helicase Origin of replication 5’ 3’ 3’ DNA Replication RNA primer Helicase Helicase 5’ 3’ 3’ 3’ Origin of replication 3’ The unconnected fragments (circled below) in the lagging strand are called Okazaki fragments, named after the Japanese couple who discovered them Reiji and Tsuneko Okazaki Tsuneko and her husband Reiji in the lab (1950s)  Died ofleukemiain 1975 at age 44 Was exposed to radiation after atomic bombing of Hiroshima Continued the research after Reiji passed away Still alive today Active research in Japan Image source: https://geneticsunzipped.com/news/2019/3/28/picking-up-the-pieces-tsuneko-okazaki A B D C Video Question In the lagging strand shown, which Okazaki fragment was made first? A. A B. B C. C D. D E. Not enough info to tell Origin of replication is here (away from fork)! DNA Replication primer 3’ Helicase Helicase 5’ 3’ 3’ 3’ Origin of replication DNA pol 1 Open DNA molecule Hold open Add primer/build new DNA molecule → Remove RNA primers DNA Replication 3’ Helicase Helicase 3’ 3’ 3’ 5’ Origin of replication Open DNA molecule Hold open Add primer/build new DNA molecule Remove primers → Fill in empty spots DNA pol 1 DNA Replication 3’ Helicase Helicase 3’ 3’ 3’ Open DNA molecule Hold open Add primer/build new DNA molecule Remove primers Fill in empty spots → Join backbones together 5’ Ligase Origin of replication DNA Replication 3’ Helicase Helicase 3’ 3’ 3’ Open DNA molecule Hold open Add primer/build new DNA molecule Remove primers Fill in empty spots  Join backbones together 5’ Origin of replication DONE! Today’s LOs Identify the 5’ and 3’ ends of a nucleotide on a DNA strand State which end (5' or 3') of a DNA strand that new nucleotides are always added to Explain how the definition of antiparallel is reflected in DNA structure Given that DNA is a double-stranded helix, state how many strands of DNA are in a single DNA molecule Describe the process of semiconservative DNA replication Define the origin of replication, the replication bubble, and the replication fork See Blackboard for a complete list of LOs State the functions of key proteins involved in DNA replication, including key enzymes and binding proteins Predict the consequence if a key protein involved in DNA replication were mutated or could not function Explain the process of DNA replication in eukaryotes Differentiate between leading strand and lagging strand Explain why DNA polymerase and primase must lay nucleotides in the 5’ →3' direction Explain why DNA polymerase needs a primer, but primase does not Describe the process of DNA replication in bacteria (i.e. circular vs linear DNA replication) Relate the role of telomeres to the shortening of linear chromosomes during each round of replication DNA Replication Recorded Lecture, Part 3 Today’s LOs Identify the 5’ and 3’ ends of a nucleotide on a DNA strand State which end (5' or 3') of a DNA strand that new nucleotides are always added to Explain how the definition of antiparallel is reflected in DNA structure Given that DNA is a double-stranded helix, state how many strands of DNA are in a single DNA molecule Describe the process of semiconservative DNA replication Define the origin of replication, the replication bubble, and the replication fork See Blackboard for a complete list of LOs State the functions of key proteins involved in DNA replication, including key enzymes and binding proteins Predict the consequence if a key protein involved in DNA replication were mutated or could not function Explain the process of DNA replication in eukaryotes Differentiate between leading strand and lagging strand Explain why DNA polymerase and primase must lay nucleotides in the 5’ → 3' direction Explain why DNA polymerase needs a primer, but primase does not Describe the process of DNA replication in bacteria (i.e. circular vs linear DNA replication) Relate the role of telomeres to the shortening of linear chromosomes during each round of replication Circular DNA Replication If circular, only one replication bubble; finished when ends meet Bacterial DNA Linear DNA Replication linear… helicase unwinds to the end chromosome END Images from video: https://youtu.be/wf6QiIlGxSg Linear DNA Replication END DNA pol 1 Remove the RNA & fill in with DNA Linear DNA Replication Site 1 Site 2 Site 3 Video Question Keeping in mind the rules of DNA polymerase, which cannot be “filled” in with new DNA? A. Site 1 B. Site 2 C. Site 3 D. All sites can be filled in DNA polymerase Ends do not get replicated in linear DNA (eukaryotes) This means DNA is not ALL replicated in eukaryotic cell division!! Linear chromosomes will become progressively shorter with each replication Chromosomes are protected from losing vital DNA by putting noncoding DNA at the end (telomeres) Telomeres are stained in yellow. Telomeres The ends of linear chromosomes are “capped” with repeated, non-coding segments of DNA (in humans: TTAGGGTTAGGG…). These DNA segments (and associated protective proteins) are called telomeres For most cell types in your body, the telomeres shorten each time a cell divides Telomeres are stained in yellow. Telomeres When telomeres become too short, this prompts cells to enter senescence—they will no longer divide Currently, people suspect that telomere shortening contributes to aging Some cells contain a protein called telomerase that maintains the lengths of telomeres during DNA replication Some types of cancer cells increase production of telomerase to support their uncontrolled growth Key Players Key players What is it? Description/Role in DNA replication process origin of replication nucleotides Sequence on DNA where helicase binds helicase enzyme Unwinds and opens DNA, creating replication bubble & replication forks single-stranded binding proteins protein Stabilizes single-stranded DNA to help hold replication bubble open A type of RNA polymerase primase enzyme Lays down primer made of RNA nucleotides primer nucleotides A set of RNA nucleotides for DNA pol begin building new DNA from DNA polymerase III enzyme Adds DNA nucleotides to free 3’ ends, beginning at primer DNA polymerase I enzyme Removes primer and replaces with DNA nucleotides DNA ligase enzyme Joins backbones together after DNA pol I replaces primer with DNA nucleotides topoisomerase enzyme Breaks, twists, and re-joins DNA ahead of replication fork to relieve strain caused by helicase unwinding DNA at fork Today’s LOs  Identify the 5’ and 3’ ends of a nucleotide on a DNA strand State which end (5' or 3') of a DNA strand that new nucleotides are always added to Explain how the definition of antiparallel is reflected in DNA structure Given that DNA is a double-stranded helix, state how many strands of DNA are in a single DNA molecule Describe the process of semiconservative DNA replication Define the origin of replication, the replication bubble, and the replication fork See Blackboard for a complete list of LOs State the functions of key proteins involved in DNA replication, including key enzymes and binding proteins Predict the consequence if a key protein involved in DNA replication were mutated or could not function Explain the process of DNA replication in eukaryotes Differentiate between leading strand and lagging strand Explain why DNA polymerase and primase must lay nucleotides in the 5’ → 3' direction Explain why DNA polymerase needs a primer, but primase does not Describe the process of DNA replication in bacteria (i.e. circular vs linear DNA replication) Relate the role of telomeres to the shortening of linear chromosomes during each round of replication DNA Replication Class Content Upcoming Due Dates Grace : Tuesday/Thursday 9:30-10:45am in Davidson 207 Stephen : Thursday/Friday 3:00-4:15pm in Life Science 130 Kylie : Monday/Wednesday 8:30-9:45am Humanities 122 Philip : Tuesday 4:30-5:45pm BAB 232 Philip : Tuesday 7:30-8:45pm BAB 232 Week 10 Reading Assignment Due Sunday by 11:59 PM Quiz 5 (DNA replication) in class Tuesday Due Tuesday at 5 PM Central dogma assignment Central dogma video lectures Due Thursday at 5 PM Prokaryote gene expression assignment Prokaryote gene expression video lectures Today’s LOs Identify the 5’ and 3’ ends of a nucleotide on a DNA strand State which end (5' or 3') of a DNA strand that new nucleotides are always added to Explain how the definition of antiparallel is reflected in DNA structure Given that DNA is a double-stranded helix, state how many strands of DNA are in a single DNA molecule Describe the process of semiconservative DNA replication Define the origin of replication, the replication bubble, and the replication fork See Blackboard for a complete list of LOs  State the functions of key proteins involved in DNA replication, including key enzymes and binding proteins Predict the consequence if a key protein involved in DNA replication were mutated or could not function Explain the process of DNA replication in eukaryotes Differentiate between leading strand and lagging strand Explain why DNA polymerase and primase must lay nucleotides in the 5’ → 3' direction Explain why DNA polymerase needs a primer, but primase does not Describe the process of DNA replication in bacteria (i.e. circular vs linear DNA replication) Relate the role of telomeres to the shortening of linear chromosomes during each round of replication DNA structure Which answer choice shows the correctly numbered DNA ends? A. 2’ ; B: 3’ B. 3’ ; B: 5’ C. 4’ ; B: 2’ D. 5’ ; B: 3’ Sugar-phosphate backbone (on blue background) Nucleotide 5C 5C 3C 3C Phosphate group Sugar (pentose) Nitrogenous base 5 end 3 end A B Pre-class Assignment Check DNA structure If a new nucleotide is added to grow this strand, which end is it added on to? A. Can be both 5 end 3 end 5C 5C 3C 3C ? ? OH Pre-class Assignment Check Image: www.news-medical.net Each new DNA molecule is composed of ½ original template strand (in blue below) and ½ newly synthesized strand (in cream below) What if one of these daughter strands replicated again? What would the “grand”daughter strand look like? Which of the following diagrams illustrates what you would see if you labeled “new” DNA over two cycles of DNA replication? A. A B. B C. C (A) Parent strands Daughter strands Original strands Labelled strands Granddaughter strands All synthesis of daughter & grand-daughter strands uses labelled strands. Labelled strands in daughter strands will stay labelled in grand-daughter strands. (B) (C) This is why DNA replication is called “semiconservative.” The daughter molecule is composed of ½ parent strand, ½ new strand in each round. After a second round of copying, one of the molecules would contain only “new” DNA. Further Explanation If a cell is “fed” labeled nucleotides during S phase so that new DNA can be visualized in blue, which of the following shows how a chromosome in this cell would look in M phase? A. A B. B C. C D. D D Recall that each sister chromatid is composed of 2 strands of DNA—one old and one new  Key players What is it? Description/Role in DNA replication process origin of replication nucleotides Sequence on DNA where helicase binds helicase enzyme Unwinds and opens DNA, creating replication bubble & replication forks single-stranded binding proteins protein Stabilizes single-stranded DNA to help hold replication bubble open A type of RNA polymerase primase enzyme Lays down primer made of RNA nucleotides primer nucleotides A set of RNA nucleotides for DNA pol begin building new DNA from DNA polymerase III enzyme Adds DNA nucleotides to free 3’ ends, beginning at primer DNA polymerase I enzyme Removes primer and replaces with DNA nucleotides DNA ligase enzyme Joins backbones together after DNA pol I replaces primer with DNA nucleotides topoisomerase enzyme Breaks, twists, and re-joins DNA ahead of replication fork to relieve strain caused by helicase unwinding DNA at fork LO: State the functions of key proteins involved in DNA replication, including key enzymes and binding proteins Key players Consequence No new DNA strand is synthesized, no replication bubble The replication bubble is unstable and continually collapses back into DNA helix There is a replication bubble, but no primer on the open DNA and no DNA is synthesized There is a replication bubble and primers on the open DNA, but no DNA is synthesized Fragments of new DNA are unconnected to each other DNA replication stops due to tangles and due to appearance of single and double-stranded breaks Use the word bank below to match the key player with the consequence if that player were to malfunction WORD BANK (not all will be used):  DNA ligase DNA polymerase III DNA polymerase I Helicase Origin of replication Primase Primer Single-stranded binding proteins topoisomerase helicase (or origin!) single-stranded binding proteins primase DNA polymerase III DNA ligase topoisomerase In the lagging strand shown, which Okazaki fragment was made last? A. A B. B C. C D. D A B D C Most recently made fragment is closest to the replication fork Fragment made first is closest to origin of replication / away from fork 3’ 3’ Helicase Helicase Origin of replication 5’ 3’ 3’ Leading vs Lagging Strands Carcinomas can use our knowledge of replication to treat disease. For example… Carcinomas contain cells that rapidly replicate DNA & divide Doxorubicin (Dox) is used to treat carcinomas Dox blocks topoisomerase activity to stop DNA replication (which will, hopefully, kill the cell) Carcinomas If DOX stops topoisomerase from completing its function, which of the following would you expect? A. There are replication bubbles, but no primers are found on the open DNA B. There are replication bubbles, but only primers are found on the open DNA C. DNA is replicated with its complementary base-pairs, but there are DNA fragments that appear to be unconnected by the backbone D. There are replication bubbles, but DNA appears to have many breaks downstream of replication fork Remember the functions of topoisomerase: break the DNA, untwist it, and re-join the strands Choice “A” occurs due to a defect of topoisomerase. Which enzymes are defective in choices “B”, “C”, “D”? Primase DNA pol 3 Ligase Herpes virus infections  Acyclovir used to treat HSV infections guanine analog Normal guanine nucleoside Acyclovir Predict how the viral DNA with the drug would look compared to the viral DNA without the drug present. A. longer DNA strand B. shorter DNA strand C. extra copies of DNA strand D. new DNA strand made If the polymerase incorporates acyclovir, then DNA pol has no 3’ end to add on to! Today’s LOs Identify the 5’ and 3’ ends of a nucleotide on a DNA strand State which end (5' or 3') of a DNA strand that new nucleotides are always added to Explain how the definition of antiparallel is reflected in DNA structure Given that DNA is a double-stranded helix, state how many strands of DNA are in a single DNA molecule Describe the process of semiconservative DNA replication Define the origin of replication, the replication bubble, and the replication fork See Blackboard for a complete list of LOs State the functions of key proteins involved in DNA replication, including key enzymes and binding proteins Predict the consequence if a key protein involved in DNA replication were mutated or could not function Explain the process of DNA replication in eukaryotes Differentiate between leading strand and lagging strand Explain why DNA polymerase and primase must lay nucleotides in the 5’ → 3' direction Explain why DNA polymerase needs a primer, but primase does not Describe the process of DNA replication in bacteria (i.e. circular vs linear DNA replication) Relate the role of telomeres to the shortening of linear chromosomes during each round of replication