BMB 462 Lecture 23 Outline of Last Lecture I. Models for DNA ReplicationII. Properties of DNA ReplicationIII. Requirements for in vitro synthesisIV. Comparing the DNA polymerasesOutline of Current Lecture I. Initiationa. Elements and Proteins involvedb. Processc. RegulationII. Elongationa. Proteins involvedb. Leading strand vs. Lagging strandc. ProcessCurrent LectureConcepts to remembers from previous courses/lectures:-I. The stages of replication:a. Initiationb. Elongationc. Terminationi. These will be discussed in the context of E. coliII. Initiationa. Elements and Proteins involvedi. Initiation begins at a certain point in the DNA, the origin. 1. OriC is the single origin on E. coli chromosome. It stands for “originin the chromosome”.2. Sometimes E. coli also has plasmids, which contain their own origin of replication, named with a different letter.ii. The origin contains 2 different elements.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.1. DUE = DNA unwinding elementa. The DUE is composed of three 13bp tandem repeats. The DUE regions are AT rich, which allows the DNA to unwind easily (because AT contains fewer H bonds than GC).b. A consensus sequence is the most common bases found at sites that interact with a specific protein. They offer context for the protein.2. DNA binding sitesa. 9bp long, and are composed of R sites and I sites.i. 5 R-sites: Bind DNA in its ATP and ADP forms. DnaA-ATP and DnaA-ADPii. 3 I-sites: Bind DnaA-ATPiii. Both sites have to be bound by DnaA-ATP for replication to start.1. A lot of ATP indicates there's a lot of energy in the cell, so there is enough energy for replication.2. A higher presence of DnaA-ADP indicates there is insufficient energy to undergo replication.iv. R sites have a higher affinity for DnaA-ATP than I sites. This ensures that both sites have bound DnaA-ATP before replication begins. The R-sites are then bound by DnaA before I-sites (both the ATP and ADP bound forms). The I-sites only get filled when DnaA-ATP is in high concentrations. When DnaA-ATP is in high concentrations, this also meansR-sites are more likely to be filled with that than with ADP-bound form.v. The first 3 bind ATP, so they are associated with a family of AAA+ ATPases.1. ATPases are associated with diverse cellular activities: they can bind both ATP and ADP. Usually the ATP-bound is the active form. (The ATPases can do something when bound to ATP that the ADP-bound form can't do, so they use ATP to switch their behaviour).3. DnaA recognizes the origin and unwinds the DNA duplex at a pointin the origin.4. DnaB, the helicase, does further unwinding to create a replication bubble.5. DnaC helps load DnaB onto the double helix.6. Primase adds the primer.7. The Single-stranded DNA-binding protein (SSB) protects the single stranded DNA.8. Topoisomerase unwinds supercoiling.9. Dam Methylase methylates the origin; the origin has to be fully methylated for replication to occur.a. After a round of replication, only the old strand will be methylated and the new will not. The cell waits for both strands to be methylated again before beginning another round of replication.b. Processi. Binding DNA in the ATP-bound form to the R and I sites leads to supercoiling.ii. DnaC helps load the DNA onto DnaB.iii. 8 DnaA-ATP bind to R/I sites --> Unwind DUEiv. DnaC-ATP loads 2 DnaB helicases onto separated strands.1. DnaB: 5'-3' helicase, unwinds DNA.v. 8 DnaA-ATP units bind to the R and I sites, and unwind the DUE. This introduces a positive writhe, which leads to a negative twist in the region.The AT rich region unwinds fairly easily. The DnaB helicase is then loaded on the small piece of unwound DNA with the help of ATP-bound DnaC and further unwinds DNA. The DnaC is then released in its ADP form.1. The DnaC loads two DnaB helicases onto the separated strands in the origin. This creates to replication forks traveling in opposite directions.vi. DnaB helicase is a 5'-3' helicase that unwinds DNA.vii. As the helicase continues to unwind the DNA, it starts supercoiling in front of the replication fork. This is where the DNA gyrase becomes useful; it relieves the supercoiling ahead of the replication fork.viii. In Summary, DNA has to be unwound for replication to start.1. DnaA-binding introduces positive writhe. That leads to negative twist for unwinding.2. The helicase DnaB further unwinds the DNA (introduces more negative twist, resulting in more positive Wr ahead of the replication fork, which is then when gyrase relieves the supercoiling).c. Regulationi. 2 Steps of Regulation:1. The R/I sites - with the DnaATP switch.2. Methylation of the OriC by DAM methylase - it methylates adenosine (creating 6N-methyladenosine).a. DAM methylase recognizes GATC sequences, which are highly enriched in the origin, so it preferentially methylatesthe origin. b. Immediately after replication, the OriC is only hemi-methylated (only the old, parental strand is methylated) sothe cell can't immediately reenter replication.i. The hemi-methylated origin is sequestered at the plasma membrane; it needs to be relieved in order for it to be fully methylated.ii. Methylation essentially acts as a timer.III. Elongationa. Proteins involvedi. There are 2 helicases bound in the replication bubble, one on each strand, running in the 5'-3' direction.ii. A DNA gyrase sits in front of each replication fork and unwinds the DNA supercoiling.iii. The primase (DnaG) adds short RNA primers to the leading and lagging strands of the replicating DNA.iv. The SSB protects unwound DNA from attack by nucleophiles.1. Single stranded DNA is far more susceptible to cleavage than double stranded DNA.b. Leading strand vs. Lagging strandi. Leading Strand Elongation1. DNA has 2 replication forks and both replications occur at the same time. However, the new nucleotides still have to be added tothe 3' end, which makes replication at both places. 2. The leading strand is synthesized continuously. As DNA is unwound, nucleotides can immediately be added to the 3' end.ii. Lagging Strand Elongation1. Because of the way the lagging strand runs, DNA replication here is discontinuous.2. Steps of Lagging Strand Elongationa. A new primer is added.b. A new clamp is added.c. The polymerase core is switched to the new clamp, and little Okazaki fragments are formed.i. The fragments will later be connected together.c. Processi. DNA
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