DNA Replication
PCR
Amplify the DNA template • Heat the DNA molecule to around 90-95C to separate the 2 strands • In the presence of primer, we prime DNA polymerase then dNTPs are the building blocks you could replicate or rebuild the complement strand. • Repeat this cycle many times then you end up with exponentially replicated DNA template.
Recombination
Create diversity Repair damage Repair Disruption
Other protein at fork along with DNA polypermase
DNA clamp (PCNA, proliferating cell nuclear antigen); ssDNA binding protein clamp loader (replication factors)
MCM is a
DNA helicases are motor proteins which can unzip double-stranded DNA.
DNA Primase
DNA primase leaves a RNA primer that provides a base-paired 3' end as a starting point for DNA polymerase
Speed vs. Error Rate
DNA replication is among the most rapid and accurate chemical reactions RNA transcription is much less accurate Somatic cells vs. germ-line cells
Leading Strand
On the leading strand, RNA priming is needed only once: at the origin. • Leading strand grows 5' to 3' continuously
Continuity vs. adaptation
Produce exact copies most of the time Change the script occasionally • Environment can change and to survive alteration to the script can occur to ensure survival
Repair
There are reasons that there DNA replication goes through a slightly different process to repair damage. However, not all the DNA damages can be repaired accurately. The cell will always leave some errors here and there. Maintaining the genome integrity is important and also genome integrity ensures that replication is as accurate as possible.
Lagging Strand
on the lagging strand, RNA primers are needed every 200 nt or so • Lagging strand also grows 5' to 3', but in small fragments (~200nt), the Okazaki fragments, that are later stitched together o a specific endonuclease (Flap endonuclease 1) is responsible for removing the RNA primer. o Then the DNA polymerase will fill that gap until it touches the end of the previous strand. Lastly, the DNA ligase will seal that gap. o DNA ligase is the enzyme that uses the energy of hydrolyzed ATP to connect to two strands. First the Dna ligase will add the high energy ATP on the 5' end to form a high energy complex then the ligation reaction will fill the gap.
Two Domains
1 is polymerization domain(monitors base pairing-only when the match is correst will the enzyme catalyze the reaction) other domain is a proofreading domain. The proofreading domain has 3'-5' exonuclease activities. So that exonuclease activity would cleave the mismatch nucleotides since it is not paired correctly w/the complement strand. You will have much bigger freedom of movement. This way with the proofreading function of the polymerase, it increases the fidelity to roughly 1/10,000,000 nucleotides copied.
Strand-directed mismatch repair
So the mistakes that escape the proofreading process will be caught with strand-directed mismatch repair. So in this mechanism, specific enzymes, MotS & MotL will find the mismatched site and bind to it and then the newly synthesized DNA normally has nicks in the new region xso that other units of mismatch repair will find the nicks and determines which strands are actually the new strands and then remove that newly synthesized strand so that the mismatched base is removed. Then the gap will be filled up by DNA polymerase.
DNA Replication is Semi-Conservatice
Each half of the parental molecule can serve as template for the synthesis of new DNA strand, and the new double stranded DNA molecule has one new strand and one old strand. this is called semi-conservative replication. Template: single strand DNA Building blocks: dNTPs (deoxyribose triphosphates) Machine: DNA polymerase
Two types of information
Genetic and epigenetic
DNA Replication in Vivo Problems
Genome consist of long, thin threaded DNA molecules 3,000,000,000bp, 2 m long Tight and complex chromatin structure. mammalian nucleus ~ 10 µm Unwind a very stable DNA double helix Speed and error
MCM mechanism
Helicase binds to the DNA Using the energy from hydrolyzing ATP, it pushes the other strand away.
Telomerase Mechanism
Telomerase binds to template strand Telomerase adds additional telomere repeats to template strand Completion of lagginig strand by DNA polymerase
MCM complex is
The MCM complex consisting of this protein and MCM2, 6 and 7 proteins possesses DNA helicase activity, and may act as a DNA unwinding enzyme.
DNA Replication purpose
The sole purpose of DNA replication is to generate identical DNA molecules, as they are the blueprint that makes life possible.
Replication fidelity
The sole purpose of DNA replication is to generate identical DNA sequences, as they are the blueprint that makes life possible. Error rate of DNA polymerization = 1/100,000 bases Total error per cell doubling = 3,000,000,000/100,000 = 30,000
Toposiomerase
Unwinds the two strands of role to relax the coil ahead. o Absence of this proteins leads to tension in the replication fork and eventually the DNA will not turn thus halting replication
Need a primer
cannot start de novo synthesis of a DNA chain.
• DNA polyermase
catalyzes the addition of nucleotides only on to the 3 end. Therefore it is synthesized 5-3. Large protein that has a speed of 100`1000 nucleotides per second
DNA Polymerase Proofreads
distinct domain with 3'- to 5'- exonuclease activity cleaves mismatched nucleotide that "wandered" into the editing site Increase fidelity to 1 error per 10,000,000 nt copied The DNA is synthesized from 5' to 3', so 3' - 5' exonuclease activity is like chew back the strand. During the chain extension, if DNA polymerase detect an mispaired base on the 3' end of the extending DNA strand, the polymerase will remove the mismatched nucleotide using 3' - 5' exonuclease activity.
DNA synthesis can only occurs in the 5-3 direction because
o Deoxynucleoside triphosphates are the only precursor for DNA synthesis reaction. o Base pairing select the complement dNTP to be added to the 3'-end; o linkage is formed by 3'-OH attacking alpha-phosphate; o hydrolysis of pyrophosphate ensure irreversible reaction. • Negative G, therefore going the other direction is highly unfavorable
DNA Replication Start
o Orc binds to post-mitotic double-strand DNA o ORC recruits other proteins to DNA replication o MCM(DNA helicase) unwinds the DNA o DNA polymerase copy strands
ORC stands for
origin of replication complex
Strand-directed mismatch repair catches mistakes escape proof reading
repair catches mistakes escape proof reading
DNA is polarized
• A single strand oligonucleotide consist of nucleotides connected through the 3-5 phosphodiester bond
MCM stands for
MCM: mini-chromosome maintenance proteins
4 Key points
Make single-stranded DNA with ORC and DNA helicase Add RNA primers with DNA primase Asymmetric DNA synthesis by DNA polymerase at the replication fork stitch DNA fragments together
DNA is replicated during
Mitosis -Maintenance -repair of damaged tissue -Embroyonic Development Meiosis Germ-line cells
DNA Is replicated during
Mitosis Meiosis
How many origins of replication in Eukaryotes
Multiple
Strand-directed mismatch repair enzymes
MutS and MutL
DNA is the
Permanent carrier of information
The challenges of copying DNA and keeping genetic information
Produce exact copies Speed vs. accuracy Repair Recombination
Telomerase
• Maintains the length of the genome o Expressed in stem cells, such as embroyonic stem cells, which proliferate indefinetly o It is turned off in most somatic cells e.g epithelia cells o Telomere function as a molecular timer add repetitive sequences on the end of the chromosome. That repetitive sequence is called a telomere.
MCM
• Mini-chromosome maintenance proteins (MCM) are essential for the initiation of eukaryotic genome replication. The hexameric protein complex formed by MCM proteins is a key component of the pre-replication complex (pre_RC) and may be involved in the formation of replication forks and in the recruitment of other DNA replication related proteins.
Life replicate Itself
• One of the fundamental properties of life is self-replication • A cell is the basic self-sustainable unit of life • When a cell replicates, it replicates the cellular machinery and the information that is needed to direct its future replication • Therefore cell replication is fundamentally a replication of information
ORC bindings cause
• The ORC binds to the host mitotic double strand DNA and recruit other proteins prior to the new DNA replication including the DNA helicases. The DNA helicases unwind the DNA to produce a single strand DNA template and then DNA polymerase copies the strand. This is the first step that you need to find the origin of replication.
Why we need telomeres
As the replication fork approaches the end of a chromosome;althought the leading strand cab ne replicated all the way to the chromosome tip, the lagging strand cannot. WHen the final RNA primer on the lagging strand is removed, there is not way to replcae it. We solve this by the use of telomerase
Error Rate for high fidelity
5'-3' polymerization 1 error per 100,000 nucleotides Proof-reading Reduce the error rate to 1/100 Strand-directed mismatch repair Reduce the error rate to 1/100 Total 1 error per 1,000,000,000 nucleotides
Three steps that give rise to high-fidelity DNA synthesis
5'-3' polymerization Proof-reading Strand-directed mismatch repair
The basic properties of DNA enable accurate replication
Base pairing enable the replication of complement DNA strand • When the structure of the DNA double helix model was proposed in in the 1950's by Crick, they noted that the specific base pairing would suggest a possible mechanism for accurate copying of the genetic info. In DNA molecules, which only contains A, T, C, G bases, adenine always pairs with thymine; guanine always pairs with cytosine. Therefor an unpaired base in a single stranded DNA can choose among the 4 precursors to assemble a complement DNA strand. This mechanism enables the accurate replication
DNA Replication in Eukaryotes is
Complicated because it has a larger genome and contains non-coding regions
Bacterial replication
DNA circular and naked Smaller One origin of replication
