BMB 462 Lecture 24 Outline of Last Lecture I. Initiationa. Elements and Proteins involvedb. Processc. RegulationII. Elongationa. Proteins involvedb. Leading strand vs. Lagging strandc. ProcessOutline of Current Lecture I. Continuing Elongationa. Reviewb. Lagging strand Primer removal and Nick sealingII. TerminationIII. Chromosomal SeparationIV. Eukaryotic DNA Replicationa. Complications Replicating Eukaryotic Chromosome EndsV. The Central DogmaVI. Polymerase InhibitorsCurrent LectureConcepts to remembers from previous courses/lectures:-I. Continuing Elongationa. Reviewi. Replication has to happen in both strandsii. Polymerase always elongates 5'-3' which makes it move along the parental strand 3'-5'.iii. The parental leading strand moves into the replication fork 3'-5' so that the new leading strand can be synthesized 5'-3'.iv. The parental lagging strand enters the replication fork 5'-3'.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.b. Lagging strand Primer removal and Nick sealingi. After the Okazaki fragment has been made on the lagging strand, there is still a primer to remove and a nick in the sequence to seal.1. That's where Polymerase I come into play. DNA polymerase I removes the RNA primer with its 5' - 3' exonuclease activity. It then fills in and finishes the strand.ii. All that's left is sealing the Nick - The nick between the Okazaki fragments is sealed by ligase.iii. Why use a RNA primer instead of a DNA primer? 1. One idea: the addition of the first few nucleotides is very error prone. So it's better to make an RNA primer and remove it than to start with a DNA primer.c. 5’-3’ Exonuclease in DNA Polymerase Ii. The need for the RNA primer to reduce nucleotide error is the reason the cell has the Polymerase I. It has an extra active site for 5'-3' exonuclease activity in a separate domain, which the polymerase III does not have.d. DNA Ligasei. The cell has an adenosine moiety that is used to activate the nick.ii. The enzyme gets adenylated - an adenyl moiety is added to a lysine residue in the active site on the enzyme - and then the adenyl is transferred to the 5' phosphate group in the nick. The 5' phosphate activated by the addition of adenylate and the 3' OH can now attack the 5' Phosphate. The adenyl then becomes a good leaving group and is released. The nick has been sealed.iii. The AMP (adenylate) comes from NAD+ in bacteria and ATP in some eukaryotesII. Terminationa. Termination occurs so that the two replication forks don't run into each other. b. There is a termination region (aka terminator) in the single chromosome of E. coli, which functions as a one directional trap.i. The polymerase follows the replication fork and enters the terminator, where it gets trapped and can no longer go back.1. There are usually multiple termination regions, so that if one fails there is another to stop polymerase progression.c. The terminator regions are bound by the protein Tus (Terminus utilization substance). If Tus forms a complex with Ter, it stops the polymerase.i. When the replication fork encounters the Ter-Tus complex, it gets trappedand halts.III. Chromosomal Separationa. After replication the two chromosomes - the old and the new - are entangled. (Atthis point they are called catenated chromosomes).i. The topologically linked chromosomes have to be disentangled by topoisomerase I, then cells can divide.1. The topoisomerase functions by breaking the backbone of the double helix and disentangling the strands.2. Topoisomerase II is used, because both strands in the double helix need to be cut in order to fully disentangle both chromosomes.IV. Eukaryotic DNA Replicationa. Complexity of Eukaryotic Replicationi. In vivo synthesis always happens on both strands. 1. If you do PCR, you replicate one strand at a time.ii. Termination has to occur at the right time and at the right site in vivo (while PCR simply stops when it runs out of template DNA to follow).iii. Another complication arises from the fact that replication has to be tied to the cell cycle (which is more complex a cycle in eukaryotes).b. The Cell Cyclei. One reason the cell cycle is more complex in eukaryotes than prokaryotesis the presence of the nucleus.1. The Nucleus has to break down in order for the chromosomes to separate and funnel to each end of the dividing cell.2. Eukaryotes also have more than 1 chromosome.a. Replication must then be coordinated so that all chromosomes are replicated at the same time. The cell must also complete the replication of each chromosome before it is divided.c. Control of Initiationi. Initiation is regulated by cyclins and cyclin dependent kinases.1. These coordinate when each step of the cell cycle happens.2. These factors are called cyclins because their concentration and activity fluctuates throughout the cell cycle.d. Major Proteins Involvedi. The next step is regulated by the helicase MCM2-7, which is loaded onto the chromosome by the ORC (Origin Recognition Complex)1. In eukaryotes, because the chromosomes are so large, there are multiple origins. Thus replication starts at many origins at the same time.e. Complications Replicating Eukaryotic Chromosome Endsi. One of the main problems of replication comes at the end of the chromosomes.1. Eukaryotes use structures called telomeresa. The leading strand can just be synthesized 5'-3' to the end of the chromosome.b. The lagging strand, however, eventually runs out of space to add a new primer. This leaves a portion of single stranded DNA, which is highly vulnerable to attack from ssDNA.i. This often results in the lagging strand becoming slightly smaller with each replication. The cell tries to counter act this but it is a problem.ii. Telomeres - To prevent the ends of the lagging strand from getting attacked, the cell uses structures called telomeres, which protect the ends.1. The ends of the chromosomes contain repetitive DNA sequences that don't code for a gene. This one way of ensuring, if those sections are lost, the cell does not lose information.iii. Telomere Extension - Even with the aid of telomeres, the chromosomes get shorter with each replication (this can be used to tell the age of a cell-senescence - the older a cell and its chromosomes are, the shorter they are).iv. RNA-Dependent DNA polymerase - Extension is done by telomerase, which is an RNA-dependent DNA polymerase.1. Telomerase adds the
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