2.3 - DNA replication in vivo Flashcards
What are the steps of DNA replication in order?
- Replication fork formation
- Primer binding
- Elongation
- Termination
Step 1: Replication Fork Formation: Before DNA can be replicated, the double stranded molecule must be “unzipped” into two single strands. In order to unwind DNA, these interactions between base pairs must be broken. This is performed by an enzyme known as DNA helicase. DNA helicase disrupts the hydrogen bonding between base pairs to separate the strands into a Y shape known as the replication fork. This area will be the template for replication to begin.
Step 2: Primer Binding: The leading strand is the simplest to replicate. Once the DNA strands have been separated, a primer generated by the enzyme DNA primase binds to the 3’ end of the strand. The primer always binds as the starting point for replication.
Step 3: Elongation: Enzymes called DNA polymerases are responsible for creating new strands through a process called elongation. There are five different types of known DNA polymerases in bacterial and human cells. In bacteria such as E. coli, polymerase III is the primary replication enzyme, while polymerases I, II, IV and V are responsible for error checking and repair. DNA polymerase III binds to the strand at the site of the primer and begins the process of adding new base pairs complementary to the strand during replication. In eukaryotic cells, polymerases α, δ and ε are the main polymerases involved in DNA replication. As replication proceeds along the leading strand in the 5’ to 3’ direction, the newly formed strand is continuous.
Step 4: Termination: Once both the continuous and discontinuous strands are formed, an enzyme called exonuclease removes all RNA primers from the original strands. These primers are then replaced with appropriate bases. Another exonuclease “proofreads” the newly formed DNA to check, remove and replace any errors. Another enzyme called DNA ligase joins Okazaki fragments together forming a single unified strand.
Sources: Reece, Jane B., and Neil A. Campbell. Campbell Biology. Benjamin Cummings, 2011.
How does DNA Pol proofread?
The polymerase checks whether the newly added base has paired correctly with the base in the template strand. If it is the right base, the next nucleotide is added. If not, it can back up and cut out the incorrect nucleotide and replace with the correct one.
What is an Okazaki fragment is and its role in replication?
Okazaki fragments are short sequences of DNA nucleotides that are synthesized discontinuously and are later joined together by DNA ligase during DNA replication to form lagging strands.
During DNA replication, the double helix is unwound and the complementary strands are separated by DNA helicases to form a DNA replication fork. After this fork, DNA primase and DNA polymerase come into to create a new complementary strand. Since these enzymes can only work in the 5’ to 3’ direction, the two unraveled template strands replicate in different ways. The leading strand undergoes a continuous replication process because its template strand has a 3’ to 5’ orientation, allowing the polymerase that assembles the leading strand to follow the replication fork. However, the lagging strand cannot be created continuously because its template strand has a 5’ to 3’ direction, which means that the polymerase must work backward from the replication fork. This results in periodic breaks in the creation of the lagging strand. Primase and polymerase move in the opposite direction of the fork, so these enzymes must repeatedly stop and restart as the DNA helicase separates the strands. Once the fragments are made, DNA ligase joins them into a continuous strand. The entire replication process is considered “semi-discontinuous” because one of the new strands is formed continuously while the other is not.
What are the types of non-covalent interactions that enable interactions between DNA and DNA-binding proteins?
H bonding, electrostatic interactions, hydrophobic interactions and van der Waals forces.
Further reading:
DNA-binding proteins are proteins that have DNA-binding domains and thus have a specific or general affinity for single- or double-stranded DNA. Sequence-specific DNA-binding proteins generally interact with the major groove of B-DNA, because it exposes more functional groups that identify a base pair.
Source: Pabo CO, Sauer RT (1984). “Protein-DNA recognition”. Annu. Rev. Biochem.
How is the DNA replication within the cell cycle of a bacterial?
The growth of bacteria can be modeled with four different phases: lag phase (A), log phase or exponential phase (B), stationary phase (C), and death phase (D).
During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual bacteria are maturing and not yet able to divide. During the lag phase of the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs. During the lag phase cells change very little because the cells do not immediately reproduce in a new medium.
The log phase is a period of cell doubling. The number of new bacteria appearing is proportional to the present population. If growth is not limited, doubling will continue at a constant rate so both the number of cells and the rate of population increase doubles with each consecutive time period. Exponential growth cannot continue indefinitely, however, because the medium is soon depleted of nutrients.
The stationary phase is often due to a growth-limiting factor such as the depletion of an essential nutrient, and/or the formation of an inhibitory product such as an organic acid. Stationary phase results from a situation in which growth rate and death rate are equal. The number of new cells created is limited by the growth factor and as a result the rate of cell growth matches the rate of cell death. The result is a “smooth,” horizontal linear part of the curve during the stationary phase. Mutations can occur during stationary phase.
At death phase, bacteria die. This could be caused by lack of nutrients, environmental temperature above or below the tolerance band for the species, or other injurious conditions.
Source: Case, Christine; Funke, Berdell; Tortora, Gerard. Microbiology An Introduction (Tenth Edition).
What’s the difference between DNA replication in vitro (PCR) and in vivo (in cells)?
PCR and DNA replication are two processes responsible for DNA synthesis. The enzyme used for DNA synthesis in PCR is a thermophilic DNA polymerase such as Taq polymerase while the enzyme used for DNA replication is DNA polymerase. Moreover, PCR uses DNA primers while DNA replication uses RNA primers synthesized by RNA primase.
How does telomerase work?
Contains an RNA template that allows the DNA strand to be lengthened by telomere repeats.
New segment of lagging strand can the be formed, so then the original template strand is entirely replicated.
How does DNA Ligase repair a DNA sequence?
Reseals backbone by joining 3’ and 5’ ends, catalyzes a phosphodiester bond.
What is the different between DNA Pol III and DNA Pol I?
DNA Pol I: Replaces RNA primers with DNA nucleotides.
DNA Pol III: Use the sequence on a template DNA strand to choose the right deoxyribonucleotides to link together.
What are the three problems of DNA Replication?
Separation, Creation of primers, and allowing synthesis to happen simultaneously from two template strands.
How to solve the three problems?
Helicase unwinds DNA at replication fork.
RNA polymerase - or RNA Primase makes RNA primers.
From the replication fork - synthesis occurs in the opposite directions i.e. in one direction on one strand and in the other direction on the other strand.
Why are primers necessary in DNA replication but not transcription?
RNA primers are needed to begin replication because DNA polymerase is unable to do it alone. DNA transcription does not have the same problem because RNA polymerase is capable of initiating RNA synthesis.
