lecture 2 Flashcards
describe the mechanism of replication
DNA replication is a highly regulated and complex process that ensures the accurate duplication of the genome before cell division. It involves a series of coordinated steps and enzymes that work together to produce two identical copies of the DNA molecule. Here’s an overview of the key steps involved in DNA replication:
- Initiation
Origin of Replication: DNA replication begins at specific locations on the DNA molecule called origins of replication. In prokaryotes, there is smi a single origin, while in eukaryotes, there are multiple origins along the DNA.
Helicase: The enzyme helicase unwinds the double-stranded DNA by breaking the hydrogen bonds between the two complementary strands, creating a structure known as the replication fork.
Single-Strand Binding Proteins (SSBs): As the strands are separated, SSBs bind to the single-stranded DNA to prevent it from reannealing or forming secondary structures.
Topoisomerase: To prevent the DNA ahead of the replication fork from becoming too tightly coiled (supercoiling), topoisomerase enzymes (like gyrase in bacteria) introduce temporary nicks in the DNA to relieve tension. - Primer Synthesis
Primase: The enzyme primase synthesizes a short RNA primer on the single-stranded DNA. This primer provides a 3’-OH group for the enzyme DNA polymerase to initiate the addition of new nucleotides.
In eukaryotes, the RNA primer is typically around 10 nucleotides long. - Elongation
DNA Polymerase: DNA polymerase is the enzyme responsible for synthesizing the new DNA strand by adding nucleotides complementary to the template strand. In prokaryotes, DNA polymerase III is the primary enzyme for elongation, while in eukaryotes, DNA polymerase δ (delta) and DNA polymerase ε (epsilon) are involved in leading and lagging strand synthesis, respectively.
Leading Strand Synthesis: On the leading strand, DNA synthesis proceeds continuously in the 5’ to 3’ direction because the template strand is oriented in the 3’ to 5’ direction, allowing DNA polymerase to add nucleotides continuously.
Lagging Strand Synthesis: On the lagging strand, DNA synthesis occurs discontinuously in short fragments called Okazaki fragments. This is because the lagging strand is oriented in the 5’ to 3’ direction, opposite to the direction of the replication fork movement. Each Okazaki fragment requires a new RNA primer. - Primer Removal and Gap Filling
RNase H and DNA Polymerase I (in prokaryotes): Once an Okazaki fragment is synthesized, the RNA primer must be removed. In prokaryotes, DNA polymerase I replaces the RNA primers with DNA nucleotides. In eukaryotes, the enzyme RNase H degrades the RNA primers, and DNA polymerase δ fills in the gaps.
DNA Ligase: After the RNA primers are replaced with DNA, the enzyme DNA ligase seals the nicks between the Okazaki fragments, creating a continuous DNA strand on the lagging strand. - Termination
Replication Forks Meet: In prokaryotes, replication proceeds until the replication forks meet at a termination site, or “ter” sequence, where proteins help end replication. In eukaryotes, replication continues until all replication origins are duplicated, and the forks meet.
Telomeres and Telomerase (in Eukaryotes): In eukaryotic cells, the ends of the linear chromosomes are called telomeres. The enzyme telomerase extends the telomeres, solving the problem of incomplete replication at the chromosome ends. Telomerase adds repetitive sequences to the ends of chromosomes, preventing loss of genetic material during replication. - Proofreading and Error Correction
DNA Polymerase Proofreading: DNA polymerases have 3’ to 5’ exonuclease activity, which allows them to proofread the newly synthesized DNA and remove incorrectly paired nucleotides. This greatly increases the accuracy of replication.
Mismatch Repair: After replication, any remaining errors are corrected by mismatch repair enzymes, which recognize and repair mismatched base pairs.
Summary of Key Enzymes and Proteins Involved in DNA Replication:
Helicase: Unwinds the DNA helix.
Single-Strand Binding Proteins (SSBs): Stabilize the unwound DNA strands.
Topoisomerase: Prevents DNA supercoiling.
Primase: Synthesizes RNA primers.
DNA Polymerase: Adds nucleotides to the growing DNA strand.
RNase H (eukaryotes) or DNA Polymerase I (prokaryotes): Removes RNA primers and replaces them with DNA.
DNA Ligase: Seals the gaps between Okazaki fragments.
Telomerase: Extends telomeres in eukaryotic cells.
Summary of the Mechanism:
Initiation: Unwinding of the DNA at the origin of replication.
Primer Synthesis: RNA primers are laid down to provide a starting point for DNA polymerase.
Elongation: DNA polymerase synthesizes new DNA on both the leading and lagging strands.
Primer Removal: RNA primers are replaced with DNA, and gaps between fragments are sealed.
Termination: Replication ends when all DNA is duplicated, with special mechanisms in eukaryotes to handle telomeres.
Proofreading: DNA polymerases and repair mechanisms ensure high fidelity of the replication process.
This well-coordinated process ensures that genetic information is faithfully transmitted from one generation of cells to the next.
what are Single-stranded binding proteins and why are they important?
SSB proteins are essential to prevent secondary structures forming, especially because primers may enhance the recognition between strands
Replication forks are bidirectional and what is important about their structure?
*Each replication fork therefore has an asymmetric structure:
*At the leading strand, the daughter copy of DNA is synthesised continuously.
*On the lagging strand, the daughter strand that is synthesized discontinuously, using the Okazaki fragments.
*The structure of these are maintained and created by accessory proteins which include a sliding clamp and topoisomerase.
The sliding clamp
*DNA polymerase cannot maintain contact with the DNA for more than around 50-200bp.
*A sliding clamp is used to keep DNA polymerase attached to the DNA template until it reaches some double stranded DNA.
The clamp complex is made of 3 parts:
*One of two parts of the sliding ring binds to the back of DNA polymerase
*The clamp loader binds to both parts of the sliding ring and controls its opening/closing.
how does Topoisomerase prevents catastrophe?
The separation of 2 wound strands, from each other, during replication leads to torsional stress as replication forks move along the DNA
Type I Topoisomerases: “nick” the phosphodiester bond in one strand to allow rotation.
Type II Topoisomerases: Form covalent bonds with both strands, forming a brief double-strand break, active where 2 double helixes cross one another
Where does DNA replication start?
DNA replication starts at specific locations on the DNA molecule called origins of replication. These are particular sequences in the genome where the replication process is initiated. The characteristics of the origin of replication can vary depending on whether the organism is prokaryotic or eukaryotic:
- In Prokaryotes:
Single Origin of Replication: Prokaryotic cells, such as bacteria, typically have a single, well-defined origin of replication. In Escherichia coli, for example, the origin is called oriC.
Bidirectional Replication: From the origin, replication proceeds in both directions, creating two replication forks that move away from the origin until they meet on the opposite side of the circular chromosome. - In Eukaryotes:
Multiple Origins of Replication: Eukaryotic chromosomes are much larger and more complex than prokaryotic ones, so they have multiple origins of replication along each chromosome. This allows for faster replication of the large genomes.
Initiation Complexes: Each origin is recognized by a complex of proteins called the origin recognition complex (ORC). These origins are activated at different times during the S phase of the cell cycle.
Bidirectional Replication: Similar to prokaryotes, replication proceeds bidirectionally from each origin.
they are located at either side of the centromere and usually there are 2 of them
Key Characteristics of Origins of Replication:
AT-Rich Sequences: Origins often have AT-rich regions (stretches of adenine and thymine) because AT base pairs are held together by only two hydrogen bonds, making the DNA easier to unwind compared to GC-rich regions.
Replication Licensing: In eukaryotes, origins are licensed to ensure that they are only activated once per cell cycle to prevent over-replication.
Summary:
DNA replication starts at origins of replication, which are specific sequences where the process begins. Prokaryotes have a single origin, while eukaryotes have multiple origins spread across their chromosomes, allowing efficient duplication of the genome
DNA replication: Prokaryotes v Eukaryotes
- Origin of Replication
Prokaryotes:
Single Origin: Prokaryotic DNA replication begins at a single origin of replication, such as oriC in E. coli.
Circular DNA: Prokaryotes have a circular chromosome, so replication proceeds bidirectionally from this one origin, forming two replication forks that move away from each other.
Eukaryotes:
Multiple Origins: Eukaryotic chromosomes are much larger and linear, requiring multiple origins of replication. Each chromosome has several replication origins to ensure the entire genome is copied in a timely manner.
Linear DNA: Eukaryotic DNA is organized in long, linear chromosomes, which necessitates the use of multiple replication forks. - Replication Rate
Prokaryotes:
Replication is generally faster in prokaryotes, with a rate of about 1000 nucleotides per second due to their simpler structure and smaller genome size.
Eukaryotes:
Replication is slower in eukaryotes, at around 50 nucleotides per second, partly due to the more complex chromatin structure (DNA wrapped around histones) and the larger size of the genome. - DNA Polymerases
Prokaryotes:
DNA Polymerase III: The primary enzyme for synthesizing the new DNA strand.
DNA Polymerase I: Responsible for removing RNA primers and replacing them with DNA.
Eukaryotes:
DNA Polymerase α: Initiates DNA synthesis by extending the RNA primer with a short DNA sequence.
DNA Polymerase δ: Synthesizes the lagging strand.
DNA Polymerase ε: Synthesizes the leading strand.
Eukaryotes also have other specialized DNA polymerases for specific tasks (e.g., DNA repair). - Primers and Okazaki Fragments
Prokaryotes:
RNA primers are synthesized by primase, and Okazaki fragments (on the lagging strand) are generally longer (about 1000-2000 nucleotides).
Eukaryotes:
RNA primers are also synthesized by primase, but the Okazaki fragments are shorter, typically around 100-200 nucleotides due to the more complex chromatin structure. - Replication Fork and Helicase Activity
Prokaryotes:
Helicase unwinds the DNA, and replication proceeds bidirectionally from the single origin.
Eukaryotes:
Multiple replication forks are formed simultaneously from different origins. Eukaryotic helicases and replication fork dynamics are more complex due to chromatin structure. - Topoisomerases
Prokaryotes:
DNA gyrase (a type of topoisomerase) is important in relieving supercoiling ahead of the replication fork.
Eukaryotes:
Eukaryotes also use topoisomerases to manage supercoiling and untangle DNA strands, though their regulation is more complex. - DNA Packaging
Prokaryotes:
Prokaryotic DNA is not packaged into chromatin. It is relatively naked, with no histones, and is supercoiled in the nucleoid region.
Eukaryotes:
Eukaryotic DNA is wrapped around histone proteins, forming a structure called chromatin. This packaging needs to be loosened during replication, and histones must be reassembled onto the newly synthesized DNA after replication. - Termination of Replication
Prokaryotes:
Replication terminates when the replication forks meet at specific termination sequences on the circular chromosome. Tus proteins can help in stopping replication at the termination sites.
Eukaryotes:
Eukaryotic replication ends when replication forks meet or when they reach the end of a linear chromosome. Since eukaryotic chromosomes are linear, they face the end-replication problem, which is solved by the enzyme telomerase. Telomerase extends the telomeres, the repetitive sequences at the ends of the chromosomes, preventing the loss of important genetic information. - Proofreading and Error Correction
Both Prokaryotes and Eukaryotes:
Both prokaryotic and eukaryotic DNA polymerases have proofreading abilities with 3’ to 5’ exonuclease activity to ensure high fidelity of replication.
Mismatch repair mechanisms also correct errors that escape the proofreading process.
the three steps that give rise to high fidelity DNA synthesis
1.) 5’—>3’ polymerization
2.) 3’—>5’ exonucleolytic proofreading
3.) strand directed mismatch repair
Mechanisms and Structures that prevent loss of integrity
Structures:
✓Centromere
✓Telomere
Proteins/enzymes:
✓DNA polymerase (proofreading)
✓Topoisomerase (see previous lecture)
Mechanisms:
➢DNA repair (of which there are many types)
Centromeres
*There is no specific sequence however this is more about the chromatin structure of this part of the chromosome.
*In more complex eukaryotes this can be several million bp in length
*Generally made of repeated sequences of DNA called alpha-satellite DNA.
*Provides strong attachment of chromosomes to microtubules
Ending Replication: Use of Telomeres
*Progressing replication forks “run into one another” which leads to the helicases being tagged with ubiquitin
*This leads to rapid dissociation from the forks
*DNA ligase and DNA polymerase fill in any gaps
*They must also stop at the end of a chromosome
-the telomere:
➢Composed of many tandem repeats of a short sequence (in humans this is GGGTTA)
➢Prevents the cell thinking the end of the chromosome is damaged DNA
➢Is recognised by sequence-specific DNA-binding proteins which then recruit the enzyme telomerase
Replicating Telomeres
*On the lagging strand the final RNA primer cannot be replaced with DNA by the repair polymerase because there is no primer ahead of it to provide a 3′-OH.
*Telomerase recognises the tip of the telomere in template strand and uses its endogenous primer to generate new repeats in a 5’ to 3’ direction.
*The lagging strand is replicated by previously mentioned DNA polymerases, using the extensions generated as a template.
DNA polymerase
*To generate an exact copy of the original DNA the DNA polymerase must have proofreadingability.
*DNA polymerases have 2 discrete sites; P is for polymerisation and E is for editing (right).
*The incorrectly paired nucleotide is removed by the E site, which has exonuclease activity and breaks the phosphodiester bond, enabling a new base to be added when the backbone is translocated back to the P site.
in eukaryotes there is an editing site at the DNA polymerase
-translocated means move
DNA repair
*Failure to remove incorrectly paired bases by proof reading leads to the “mismatched” pair in the DNA sequence.
-errors can be caused by damage caused by sunlight and or other things such as drugs
*This is then detected, and a repair is attempted.
*The repair mechanism depends on the type of error that has been incorporated
➢Nucleotide Excision Repair (pyrimidine dimer)
➢Base Excision Repair (deamination)
➢Mismatch Repair (replication errors
Failure of repair generates a variant in the gene sequence
DNA repair –other types of change
When depurination occurs, there is no change in the base pair sequence, however there is a deletion of a base. This cannot be repaired, instead we lose a base of the sequence
