Lesson 2: Central Dogma of Molecular Biology: Replication PDF

LESSON # 2: Central Dogma of Molecular Biology: Replication Trans Checker: De Gana Trans Makers: De Gana Ituralba Abrantes Transcriber Task Progress Links De Gana Recording Part 1 Completed Part 1 Ituralba...

LESSON # 2: Central Dogma of Molecular Biology: Replication Trans Checker: De Gana Trans Makers: De Gana Ituralba Abrantes Transcriber Task Progress Links De Gana Recording Part 1 Completed Part 1 Ituralba Part 2 (Start - 0:50:00) Completed Abrantes Part 2 (0:50:00 - End) Part 2 Completed deadline: Sept 12 ____________________________________________________________________________ Central Dogma of Molecular Biology Objectives: Discuss different proteins that come into play in replication, transcription, and translation. Examine the importance of each step. Determine the how’s and why’s. DNA Replication Semi-conservative replication ○ Half of the original template is conserved while the other strand is newly synthesized ○ Semi - half of the original strand is involved in replication ○ Conservative - that half original strand is conserved while the other strand is newly synthesized S phase or synthesis phase Initiation, Elongation, and Termination 5’ to 3’ direction ○ This is the unidirectionality of the DNA where a free hydroxyl (OH) group is at the 3’ end and a free phosphate group is at the 5’ end. DNA Replication: Initiation Initiation begins with the activity of the protein complex called ORC  LESSON # 2: Central Dogma of Molecular Biology: Replication QUESTION: Why was this called a protein complex? Because a protein complex is a congregation of proteins to make one major protein Proteins that make up a protein complex could be different from one another or could be the subunits of the protein itself Formation of the Pre-replication Complex: ORC ○ or Origin Recognition Complex ○ consists of ORC1 - ORC6 ○ binds to the origins of replication ○ during the early stages of G1, ORC travels across DNA sequences to find specific regions called the Origins of Replication where replication begins ○ then the ORC binds to the origins of replication then signals and recruits two proteins called CDC6 and CDT1 QUESTION: What makes such regions the origins of replication? Recall that the types of bonds that bind base pairs together are hydrogen bonds ○ The reason for this is because hydrogen bonds are strong enough to hold the structure of the DNA, but weak enough to be separated during DNA replication and translation processes. ○ Hydrogen bonds are used because we need a bond not too strong to be able to be cleaved and allow access to the DNA, but not too weak to allow instability. With this, such regions are considered origins of replications because these are A-T rich regions. We need Adenine and Thymine because these bases possess only 2 hydrogen bonds which makes unwinding easier. In the origins of replication, Adenine and Thymine are given importance. True or False. Are there many origins of replications in eukaryotes? True This is because eukaryotes have longer genomes than the prokaryotes. It does not make sense that in the long genetic sequence of eukaryotes, only 1  LESSON # 2: Central Dogma of Molecular Biology: Replication A-T rich region or origin of replication is present. In eukaryotes, there are multiple origins of replication per chromosome while prokaryotes only have one per chromosome. Additionally, since prokaryotes only have one origin of replication, the whole bacterial chromosome could already be considered as the replicant. Prokaryotes only have one origin of replication called ORIC. Eukaryotes have multiple origins of replication called ORI. Epigenetic modification in the DNA allows the rise of origins of replication. How does the ORC recognize that the A-T binding is already enough to be considered as the origin? Once the ORC recognizes that the A-T binding has already reached 50-70%, then it considers it as an origin of replication The ORC binds to the first origin that it recognizes Cell Division Cycle 6 (CDC6), and Chromatin Licensing and DNA Replication Factor 1 (CDT1) ○ these stabilize the ORC, and guide the MCM ○ CDC6 these stabilize the binding of the ORC and the origin of replication to prepare for the other incoming proteins which will serve as supports as this whole replicative helicase complex is so big and heavy ○ CDT1 this guides the MCM helicase going to the origin of replication Helicase is an enzyme responsible for the unzipping of the DNA strand yielding a replication fork Minichromosome Maintenance (MCM) protein complex ○ main DNA helicase involved in DNA replication ○ this is a hexameric protein or protein ring composed of MCM2 - MCM7 ○ this is the main protein responsible for the unwinding of the strands Pre-replication complex (pre-RC complex)  LESSON # 2: Central Dogma of Molecular Biology: Replication RECAP: 1. The Origin Recognition complex (ORC) travels the DNA sequence to find an A-T rich region that will serve as the Origin of Replication. 2. It will then bind to the Origin of Replication (ORI for eukaryotes; ORIC for prokaryotes). 3. Once ORC is bound with the Origin of Replication, it will recruit 2 proteins called Cell Division Cycle 6 (CDC6) and Chromatin Licensing and DNA Replication Factor 1 (CDT1) which are needed for a successful and stable helicase protein. 4. Once CDC6 and CDT1 are bound, Minichromosome Maintenance (MCM) helicase can now bind to the Origin of Replication. NOTE: The helicase is still inactive at this point. Although it has already been attached to the origin of replication, the actual process of DNA replication will not start yet. But if this is the case, why does it appear that replication has already started as early as the G1 phase? ○ This is because of the replication licensing system which acts to make sure that no section of the genome is replicated more than once for each replication cycle. ○ This is the reason why the presence of the proteins CDC6, CDT1 is important. They ensure tight control over replication all in the purpose of ensuring only one replication per cell cycle occurs. How does this one replication per cycle is ensured? ○ This is where the activation of the MCM helicase and the subsequent processes come into play.  LESSON # 2: Central Dogma of Molecular Biology: Replication Initial Part of the MCM Activation: During S phase, CDKs and DDKs phosphorylates MCM complex and phosphorylates CDC6 and CDT1; thus degrading them ○ Cyclin-dependent kinases (CDKs) and Dbf4-dependent Cdc7 kinases (DDKs) activate the MCM helicase by phosphorylating it. QUESTION: What do kinases do? They are known to attach phosphate groups to certain molecules. ○ This is still in the topic of ensuring one replication per cell cycle occurs. ○ Suppose that MCM is already activated, the CDKs and DDKs will then phosphorylate CDC6, which in turn will signal for its ubiquitination or the addition of a ubiquitin on a target protein which are proteins that are unwanted or damaged. Ubiquitination ○ Ubiquitin tag is added onto a molecule signaling the proteasome to degrade the molecules by proteolysis. ○ Ubiquitin tag signals the cell that a specific protein is damaged and must be removed. ○ This is a post-translational modification to the proteins which means that changes in the protein happen after the protein is formed.’ QUESTION: How is the unwanted protein removed by the ubiquitin? Protein tags that are bound by ubiquitin will be detected and bound by a protein complex named proteasome. This proteasome degrades proteins in a process called proteolysis in which it breaks down peptide bonds using the enzyme proteases.  LESSON # 2: Central Dogma of Molecular Biology: Replication QUESTION: Why do we need to remove CDC6 and CDT1 once MCM is activated? Why are these phosphorylated? Aren’t CDKs and DDKs only for the activation of MCM? CDC6 and CDT1 need to be removed to prevent another MCM from binding which could lead to re-initiation at the same origin of replication. MCM alone cannot invite other MCM. If CDC6 and CDT1 are kept, these will again signal MCM or helicase which in turn repeat a replication cycle which could lead to genomic instability. How would this result in genomic instability? If you have an oversupply of genes, and that specific gene is needed by the cell, then all genes will be transcribed causing the overexpression of the gene and overproduction of proteins leading to impeded cellular functions. Also, if these genes are oncogenes then these will promote uncontrollable cell division. Overproduction of these will cause unchecked cell growth ultimately leading to cancer. Additionally, we know that DNA and the cells cannot replicate forever because of the telomeres and its telomerases (end replication problem), over replication of the genes would increase the rate of degradation of these telomeres which could cause early senescence or cellular aging. What comprises the pre-RC (replication) complex? ORC Complex (ORC 1-6) CDT1 and CDC 6 MCM (2-7)  LESSON # 2: Central Dogma of Molecular Biology: Replication Is this pre-RC complex active or not active? It is still inactive What stage of the cell cycle is this complex formed? Early G1 phase Licensing of the pre-RC complex: Licensing allows for the initiation step to proceed to the downstream processes of replication, activating the MCM (helicase) and leading to the S phase ○ Kinases are involved in phosphorylation of protein complexes which opens up the protein complex allowing for the binding of other proteins. Steps in Licensing ○ 1) DDKs phosphorylates the N-terminal tail of the MCM complex The phosphorylation of the MCM complex opens up the protein allowing access for more binding sites, where further reaction would occur. ○ 2) CDKs phosphorylates Treslin and RecQ4  LESSON # 2: Central Dogma of Molecular Biology: Replication Phosphorylated Treslin and RecQ4 becomes activated; working as scaffolds (docking points) for TopBP1 ○ 3) Formation of Treslin-TopBP1 protein complex These phosphorylated docking points, recruits TopBP1 forming a complex known as Trelin-TopBP1 complex ○ 4) Treslin-TopBP1 complex recruits CDC45 and GINS proteins to form the CMG protein complex CDC45-MCM-GINS protein complex CDC45 ○ Enhances MCM activity by providing structural elements that improves its: a) interaction with DNA, b) translocation speed, and c) base pair separation activity GINS ○ Acronym for Japanese numbers “5 (ko), 1 (ichi), 2 (ni), 3 (san)” Because this protein-to-protein complex is composed of SLD-5, PSF-1, PSF-2, and PSF-3 This is the active form of the MCM helicase, the main replicative helicase in eukaryotes Responsible for breaking the hydrogen bonds of the DNA and prepare them for replication Start of the Initiation Process: 1) The active helicase (CMG complex) unwinds the DNA and cleaves hydrogen bonds to form “ssDNAs” (single-stranded DNAs) ○ It requires A LOT of energy to separate this strands to form two (2) ssDNAs 2) Single-stranded binding proteins (SSBPs) bind to the ssDNAs to protect and stabilize them ○ ssDNAs have the tendency to attach back together to form DNA to become stable = SSBPs works to prevent this re-annealing for replication process to continue ○ SSBPs also allows the cleaving of ssDNAs by endonucleases Exonucleases vs endonucleases Exonucleases begins cleavage of DNA from the outside of the DNA strand, at both ends  LESSON # 2: Central Dogma of Molecular Biology: Replication Endonucleases cleaves DNA from within the strand instead from both ends ○ SSB proteins in prokaryotes = Replication protein A (RPA) in eukaryotes QUESTION: Why aren't exonucleases utilized instead of endonucleases? In cases of nucleotide mismatch, it may occur anywhere within the DNA. As exonucleases must start cleavage of DNA from its either ends, it would require the expenditure of too much energy to cleave and reach the mismatched nucleotide, especially if the mismatched nucleotide is found in the middle. If endonucleases are utilized, cleaving of mismatch becomes more efficient as it begins at the site of the mismatch. 3) RPA (SSBPs in prokaryotes) stabilizes ssDNA and prevents them from endonucleases as it can be recognized as damaged/foreign DNA  LESSON # 2: Central Dogma of Molecular Biology: Replication ○ Endonucleases are always active and might misrecognize the ssDNA as damaged DNA 4) Separated ssDNAs opens up the “replication bubble” ○ Replication bubble is the site where replication of the DNA occurs ○ At the both ends of the replication bubble is the Y-shaped structure called “replication fork,” where DNA is constantly being unwinded 5) Rapid and constant twisting of DNA causes “supercoiling” ahead of the replication fork ○ DNA Topology: studying the changes in structure of the DNA ○ The unwinding of DNA actively introduces supercoiling which may lead to super tight DNA = twisted so much that the replication fork cannot open further. 6) Topoisomerase I and II are enzymes that acts to relieve the torsional stress (relieve the supercoiling caused by unwinding of DNA) ○ It will cut one strand of DNA by cleaving its phosphodiester bonds = relieving it of torsion then ligates it back. ○ Topoisomerase I: cuts one DNA strand and rejoins it back Can function without ATP ○ Topoisomerase II: cuts both DNA strand and ligates them back Requires ATP as it requires more energy ○ Mechanism: active sites of topoisomerase have tyrosine residues with free hydroxyl group which allows for reacting with and stealing the phosphate from DNA to form phosphotyrosine bonds Then later on will relieve for ligation back to the DNA  LESSON # 2: Central Dogma of Molecular Biology: Replication Both enzymes act on introducing positive and negative supercoiling (produced by the continuous unwinding of the replication forks) Positive Supercoiling: tightening the DNA (producing coils upon coils upon itself) Negative Supercoiling: underwinding of DNA Elongation QUESTION: Before we are able to produce a new strand, what do we need in order to start the addition of a new strand? RNA primer added by DNA primase provides necessary 3’ hydroxyl terminus for DNA polymerase DNA polymerase can only add nucleotides on existing nucleotides with free hydroxyl groups (3’) DNA primase (Eukaryotes) or DnaG (Prokaryotes) adds RNA primers to origin region ○ There are no DNA primers existing naturally (only in laboratory setting) ○ About 10-12 primer bases ○ It reads from 3’ to 5’ and synthesizes 5’ to 3’ = as DNA is antiparallel Providing free hydroxyl group = DNA polymerase becomes excited to attach nucleotides for elongation QUESTION: Why aren't there any naturally occurring DNA primers? Chemical Evolution during Prebiotic history: RNA was first formed before the occurrence of the more complex DNA, thus this print of RNA has been present since then. It can be synthesized DE NOVO: from scratch (on its own), DNA primase can create an RNA primer on its own without template compared to DNA polymerase which requires template strand and free 3’ hydroxyl for attachment of nucleotides.  LESSON # 2: Central Dogma of Molecular Biology: Replication Alerts DNA for proofreading: since it’s RNA, it alerts the cells that there is an RNA among with the DNA nucleotide bases > encouraging the DNA to proofread and eventually remove the RNA = effectively increasing DNA integrity and fidelity Prokaryotic DNA polymerases (1) DNA polymerase 3 - Additional: responsible for synthesizing new DNA strands. (2) DNA polymerase 1 - Additional: involved in DNA repair and replacing RNA primers with DNA. Eukaryotic DNA polymerases (1) DNA polymerase epsilon (Pol ε) (2) DNA polymerase delta (Pol δ) (3) DNA polymerase alpha (Pol 𝝰) QUESTION: What is the key difference between DNA Pol ε and DNA Pol δ? DNA polymerase epsilon (Pol ε) - responsible for synthesizing the leading strand DNA polymerase delta (Pol δ) - responsible for synthesizing the lagging strand What is the function of DNA Pol 𝝰? It initiates nuclear DNA synthesis and DNA repair. It also has primase activity. ○ Additional: DNA Pol 𝝰 works with primase to generate the primers necessary for DNA synthesis. It fills the gap between Okazaki fragments synthesized by DNA Pol δ ○ INCORRECT: After RNase H removes the RNA primers, DNA Pol ε fills the gaps on the leading strand, while DNA Pol δ fills the gaps on the lagging strand. From the word ‘alpha’ which means starting, DNA Pol 𝝰 is responsible for synthesizing the RNA primer for DNA replication. This primer will be then extended by DNA Pol ε and DNA Pol δ. It directly works with DNA primase to create an RNA-DNA primer hybrid at the origin of replication. After that, DNA Pol 𝝰 will be replaced by either DNA Pol ε or DNA Pol δ, with DNA Pol ε working with the leading strand while the DNA Pol δ on the lagging strand. What is the difference between leading and lagging strands? The leading strand is continuously synthesized by DNA Pol ε, while the lagging  LESSON # 2: Central Dogma of Molecular Biology: Replication strand is discontinuously synthesized by DNA Pol δ, making short fragments called the Okazaki fragments. The leading strand requires only one primer to initiate synthesis, while lagging requires multiple primers for each Okazaki fragment. The leading strand is synthesized continuously because it runs in the 3' to 5' direction relative to the replication fork, allowing DNA polymerase to add nucleotides continuously from the 5' to 3' direction. In contrast, the lagging strand runs in the 5' to 3' direction relative to the replication fork. This requires DNA polymerase to synthesize it in short fragments as it can only synthesize DNA in the 5' to 3' direction and must work backward from the replication fork. ○ Leading strand – DNA Pol ε synthesizes continuously in the same direction as the replication fork moves. This means that it adds nucleotides toward the direction where the helicase is unwinding the DNA. The direction of replication fork -> 5’ to 3’ (left to right) ○ Lagging strand – DNA Pol δ cannot synthesize continuously as it would need to add nucleotides in the opposite direction of the fork’s movement. For this reason, the lagging strand is synthesized in discontinuous fragments (Okazaki fragments), with each fragment requiring a new primer as the fork progresses. Is the replication fork bidirectional? Bidirectional because replication bubbles are formed at both ends. At each replication fork, the leading strand is synthesized towards the fork, while the lagging strand is synthesized away from the fork. Why is the synthesis of the leading strand continuous while the lagging strand is discontinuous? Leading Strand – DNA primase will first create an RNA primer of 10-12 bases. Once this primer provides a free 3’-OH group, DNA Pol ε adds nucleotides at a rate of 1000 nucleotides per second. And since the leading strand runs in the same direction as the replication fork, DNA Pol ε synthesizes nucleotides continuously. Lagging Strand – DNA primase synthesizes RNA primers as the helicase unwinds the DNA. The lagging strand is oriented in the opposite direction of the replication fork, thus, as the DNA continues to unwind, new sections of the template strand become longer and exposed. So, the DNA primase creates another primer for each exposed segment or section. DNA Pol δ adds nucleotides to these primers forming Okazaki fragments. New primers are continuously made as the DNA unwinds further, resulting in a fragmented and discontinuous synthesis of the lagging strand.  LESSON # 2: Central Dogma of Molecular Biology: Replication What do you call the fragmented DNA found in the lagging strand? Okazaki fragment – discovered by a superpower couple from Japan in the 1960s, Reiji and Tsuneko Okazaki. Since we are only replicating the DNA, how can we remove the RNA from the preparation? RNase H or ribonuclease H – cleaves off the ribonucleases or RNA primers. ○ Reads the DNA and recognizes the RNA-DNA hybrid at the beginning of the DNA. It checks both the leading and lagging strands. On the leading strand, RNase H encounters RNA-DNA hybrids formed by the RNA primer from DNA primase and the initial DNA segment from DNA Pol 𝝰. On the lagging strand, RNase H encounters the multiple primers made by the DNA primase. ○ Finds all the RNAs in the strands and removes them. DNA Pol ε (leading) and DNA Pol δ (lagging) will read the strand and decide what nucleotides to add. ○ The removed RNA earlier will be replaced by DNA. ○ Proofreading – DNA Pol checks the strand in its previous work for mismatches and corrects any errors during the replication. RNase H removes the primers in the leading and lagging strands. Pol ε adds the bases on the leading strand and DNA Pol δ adds the missing bases on the lagging strand. QUESTION: What happens to the nicks and cuts of RNase H? How are they joined together? Another protein comes into play, the DNA ligase. ○ DNA ligase – seals off the nicks done by RNase H and ligates the new strands to the leading strand also ligates the Okazaki fragments in the lagging strand to create one continuous strand.  LESSON # 2: Central Dogma of Molecular Biology: Replication As shown in the figure above, after RNase H cuts or removes the RNA primers, DNA flaps are created. ○ These flaps are then removed by a protein called FEN1. ○ RNase H and FEN1 work together: RNase H cleaves the primers that create flaps (excess), and FEN1 subsequently removes these flaps. ○ Additional: Flap endonuclease 1 (FEN1), also known as DNase IV, removes RNA and DNA 5′-flaps. It is involved in the base excision repair pathway and counteracts replication stress. Termination At the end of replication, as the DNA helicases unwind the DNA, they will eventually meet each other.  LESSON # 2: Central Dogma of Molecular Biology: Replication ○ There will be a collision (two replication forks collide) which signals an end to replication (E in the figure above) RNA primers are removed by RNase H, DNA polymerase proofreads, and DNA ligase ligates the cuts. In eukaryotic cells, there is what we call the end replication problem. ○ This problem arises because polymerase cannot replicate the very end of the chromosomes. Telomeres – protective structures at the ends of chromosomes Centromeres – the central region of the chromosome that holds the sister chromatids together. (A) The parent strand composed of two strands is separated in preparation for replication.  LESSON # 2: Central Dogma of Molecular Biology: Replication (B) & (C) The red lines are the newly synthesized sequence complementary to each parent strand. After replication, DNA primase creates RNA primers (yellow in the figure) initiating the synthesis of DNA sequence. Once replication is complete, this RNA primer will be removed by ribonuclease H (RNase H). RECAP: Rule of DNA Polymerase Read – 5’ to 3’ Add – 3’ end, elongating the DNA in 5’ to 3’ direction QUESTION: Why can't the 5' ends of the newly synthesized strand, where RNA primers were removed, be filled after the actions of RNase H? See (D & E) in the figure above DNA polymerase becomes excited when encountering the 3’ hydroxyl group because this is where it can only add nucleotides. The newly synthesized strand is now shorter than the parent or original strand because it cannot be fully filled in by DNA polymerase. In each cell replication cycle, the chromosomes or the genomic DNA become shorter and shorter. “Repetitive replication shortens the genes” QUESTION: What does the cell do to prevent this? Telomerase – the enzyme that extends the telomeres ○ Since repetitive replication shortens our chromosomes, telomerases help to lengthen the telomeres at the ends of chromosomes. ○ It protects the coding regions by adding repetitive noncoding sequences at the ends. ○ As we grow old, the function or activity of telomerase decreases, leading to the gradual shortening of telomeres. Once the telomeres become too short, they can no longer protect the ends of chromosomes, which can result in the loss of essential coding regions of DNA, also known as gene loss. Senescence or cellular aging Additional: increases the risk of age-related diseases as critical genetic information may be compromised. Aging is partly caused by free radicals or oxidants that are present if there is significant cellular stress.  LESSON # 2: Central Dogma of Molecular Biology: Replication These oxidants cause genetic damage, aberration, and cancer. What are the repeating DNA sequences of telomeres? 5′-TTAGGG-3′ Is telomerase upregulated in cancer? True or False True. Telomerase is upregulated in cancer cells. While telomerase activity decreases as we age, in cancer cells, the opposite occurs. This allows continuous cell proliferation, preventing the usual shortening of telomeres, which contributes to uncontrolled growth. Lab-grown – called immortalized because they are capable of infinite replication, thus, infinite growth. ○ Additional: Lab-grown refers to cancer cells that are cultured in the laboratory setting for research purposes (understanding cancer and developing effective treatments). QUESTION BY STUDENT: Telomerases create a T-loop structure by connecting the 3' overhang to the 5' end, forming a protective capping structure that shields DNA from degradation. Does this mechanism only apply to eukaryotes? Telomeres are only present in Metazoans or higher forms of animals, and not found in prokaryotes. This is because prokaryotes have circular DNA which lacks distinct ends. ○ In prokaryotes, the origin of replication is a single site known as OriC, while in eukaryotes there are many origins of replication. QUESTION: How many telomeres are there in a single chromatid? In a chromatid, there are 2. In a chromosome, there are 4 (2 per chromatid) QUESTION BY STUDENT: How does telomerase add nucleotides to the ends of DNA? Through reverse transcription ○ Telomerase will first read the parent or original strand (3’ end) and then will start to add RNA bases complementary to that template. ○ Imagine that telomerase has 2 arms:  LESSON # 2: Central Dogma of Molecular Biology: Replication The first arm creates an RNA complement of the parent strand. The second arm creates a DNA strand complementary to the RNA complement created by the first arm. Where does the RNA used by telomerase come from? Is it synthesized by telomerase? From the cell itself or free-floating in the cell.