Term
| genomic instability leads to |
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Definition
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Term
| How do cells duplicate their DNA without introducing errors? |
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Definition
1) DNA replication depends on DNA template mechanism
- Recognition of each nucleotide in the DNA template strand by a free complementary nucleotide, Base-pairing underlines DNA replication |
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Term
| How can the two strands act as a template for DNA polymerase if one runs 5’-3’ and the other 3’-5’? |
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Definition
| Leading and lagging strand replication are coordinated. Lagging strand synthesis occurs via short strands called Okazaki fragments. Leading strand synthesis occurs continually until elongation reaches another replication bubble |
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Term
Where does replication begin on the DNA?
What are replication origins? |
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Definition
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Term
How does a cell know when to start replication?
What controls origin firing? |
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Definition
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Term
| What are the two single, separate strands of replicating DNA called? |
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Definition
| Template and primer strands |
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Term
Is DNA replication semi-conservative, conservative or dispersive?
Meselson & Stahl, N15 and N14 |
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Definition
Semi-conservative - conservative keeps the parent strands together, and dispersive is a random combination of parent and daughter on each strand
Replication is semiconservative. Each strand of the DNA is used as a template to make a new strand. So, each replicated duplex has one old and one new strand. |
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Term
| Replication occurs along both DNA strands |
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Definition
We call the template strand that supports synthesis towards the fork the leading strand template
We call the template strand that supports synthesis away from the fork the lagging strand template - 30 million Okazaki fragments in one round of replication in humans |
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Term
| How does DNA polymerase add nucleotides to a growing daughter strand (direction, free energy) |
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Definition
DNA polymerases add nucleotides to the 3’-OH of a chain by the principle of complementation using the deoxynucleoside triphosphates as substrate and energy source.
There is release of free energy in the form of pyrophosphate and that is subsequently hydrolyzed to release two molecules of inorganic phosphate.
DNA polymerases catalyze the nucleophilic attack of the 3’-OH of a primer on the alpha-phosphate of an incoming deoxynucleoside 5’-triphosphate chosen by complementation to the template. The primer must be annealed, i.e., base paired, to the template. |
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Term
| How does DNA polymerase joining work? |
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Definition
It resembles a right hand in which palm, fingers and thumb grasp the DNA and form the active site.
The correct positioning of an incoming dNTP causes the fingers of the polymerase to tighten, thereby initiating the nucleotide addition. Release of energy causes the fingers to relax and prepare for the next round of nucleotide addition.
DNA polymerases catalyze the nucleophilic attack of the 3’-OH of the primer on the alpha phosphate of the deoxynucleoside triphosphate to liberate pyrophosphate. |
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Term
| Cells have many DNA polymerases: Prokaryotic, Eukaryotic |
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Definition
Prokaryotic (replication polymerases):
I - Essential; used in Okazaki fragment joining
III - Used for both leading and lagging strand replication
(II is used mainly is DNA repair)
Eukaryotic:
alpha - DNA replication initiation
delta - Lagging strand replication
epsilon - Leading strand replication
(Beta is used mainly in DNA repair)
(Gamma is used in mitochondrial DNA replication) |
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Term
| DNA polymerases have a high fidelity |
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Definition
The fidelity of copying DNA during replication is very high. It is only about 1 mistake in 109 nucleotides copied
The fidelity of copying DNA during replication is very high. It is only about 1 mistake in 109 nucleotides copied |
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Term
| Exonucleolytic proofreading activity of DNA polymerases |
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Definition
DNA molecules with a mismatched nucleotide at the 3’-OH end of the new strand will not be extended by the polymerase.
Most DNA polymerases possess a 3’-5’ exonuclease activity that removes mismatched bases.
The exonuclease is usually called the proofreading activity.
Note that it “goes” in the opposite direction of the polymerase. 3’-5’ direction
Most DNA polymerases possesses a separate active site that catalyzes the 3’-5’ exonucleolytic removal of mismatched bases. It is called the proofreading exonuclease. |
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Term
| The proofreading exonuclease often constitutes a second active site |
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Definition
| In the editing mode, the newly synthesized DNA transiently unpairs from the template and the polymerase undergoes a conformation change to allow editing |
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Term
| Very few errors are introduced during replication - Why? |
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Definition
5' - 3' polymerization 1 in 100K
3' - 5' exonucleolytic proofreading 1 in 100
Overall, 1 in 10M errors from DNA polymerase
Add strand-directed mismatch repair, another 1 in 100, becomes 1 in 1B errors |
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Term
| DNA Primase is required for DNA alpha polymerization |
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Definition
DNA polymerase cannot add nucleotides de novo, so RNA primer is required, about 10 nucleotides long, to provide a 3'-OH group for DNA pol alpha to begin elongation
DNA polymerases require a primer. In eukaryotes this primer is 10 nucleotides long.
Primase is an RNA polymerase.
In prokaryotes it is the DnaG protein.
In eukaryotes it is part of a complex that includes DNA polymerase alpha.
Because DNA polymerases cannot synthesize chains de novo, they require a primer. Cells have an enzyme named primase that makes a template-directed RNA primer. DNA polymerase adds to the RNA primer. |
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Term
Lagging strand synthesis
flap endonuclease |
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Definition
The RNA primer is then joined to DNA fragment forming RNA-DNA hybrid. The DNA is formed by DNA polymerase.
Lagging strand has these RNA-DNA primers after every 100-200 nucleotides.
The RNA of the penultimate Okazaki fragment is removed.
In prokaryotes the RNA primer is removed by a 5’-3’ exonuclease of Pol I.
In eukaryotes the RNA primer is removed by the flap endonuclease.
Removing RNA primers requires the action of a 5’-3’ exonuclease. DNA polymerases fill in the gap left from exonucleolytic removal, and DNA ligase seals the nick. |
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Term
Okazaki fragments are joined by
DNA Ligase I |
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Definition
DNA ligase I an essential enzyme.
DNA ligase I uses ATP and makes an AMP-intermediate at the 5’-phosphoryl of the nick. The 3’-OH at the nick then carries out a nucleophilic attack on the alpha-beta phosphodiester to join the 3’- and 5’- hydroxyls of the DNA in a phosphodiester bond.
DNA ligases recreate the phosphodiester bond by first attaching AMP to the 5’-end of the nick and catalyzing the nucleophyllic attack of the 3’-OH on the AMP linked to the 5’-phosphate. |
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Term
| A DNA helicase unwinds the DNA to open the fork |
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Definition
DNA double helix is very stable under physiological conditions. Requires a high temperature close to 100oC to open the DNA.
DNA helicases hydrolyze ATP to ADP + Pi.
They circle one of the two strands and travel along that strand unwinding the helix.
They are named for the direction they move along that strand.
Many DNA helicases are hexameric machines
DNA helicases unwind the duplex. They hydrolyze ATP. They are named for the direction they travel along the strand to which they bind. |
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Term
DNA helicases
Prokaryotic; Eukaryotic
primosome? |
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Definition
Prokaryote: DnaB
- encircles the lagging strand template unwinding the leading strand template
complexed with primase to make the primosome
Eukaryote: MCM2-7
- hexamer made up of different subunits |
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Term
Single strand binding proteins prevent the strands from rewinding
Prokaryote; Eukaryote |
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Definition
Prokaryote: ssb (single subunit)Single Stranded Binding Protein
Eukaryote: RPA (three subunits)Replication Protein A
used to prevent single-stranded regions from creating short hairpins
Single strand binding proteins prevent strands separated by helicases from reannealing. |
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Term
Sliding clamp: the Swiss army knife of DNA replication
Prokaryote; Eukaryote |
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Definition
Prokaryote: homodimer named beta
Eukaryote: homotrimer named PCNA
A sliding clamp binds replication enzymes, making DNA polymerases processive and coordinating the entry of the enzymes needed for replication |
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Term
Properties of sliding clamps?
processive; processivity factor?
CAF |
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Definition
Replicative DNA polymerases are bound to the sliding clamp. They make the polymerase processive, i.e., the polymerase remains bound to the DNA. Also called processivity factor.
Many other enzymes are also brought to the replication fork by the sliding clamp. Among these are DNA ligase I and the flap endonuclease.
Components of the mismatch repair and nucleotide excision repair systems also bind to the sliding clamp. CAF (chromatin assembly factor) is another enzyme brought to the fork by the sliding clamp. |
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Term
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Definition
Chromatin Assembly Factor
Replicative DNA polymerases are bound to the sliding clamp. They make the polymerase processive, i.e., the polymerase remains bound to the DNA. Also called processivity factor.
Many other enzymes are also brought to the replication fork by the sliding clamp. Among these are DNA ligase I and the flap endonuclease.
Components of the mismatch repair and nucleotide excision repair systems also bind to the sliding clamp. CAF (chromatin assembly factor) is another enzyme brought to the fork by the sliding clamp. |
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Term
Sliding clamps are loaded by clamp loaders
Prokaryote; Eukaryote |
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Definition
- pentameric
- hydrolyze ATP
Prokaryote: called gamma-delta
Eukaryote: called RFC (replication factor C)
Clamps probably recycle with each Okazaki fragment |
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Term
| Leading and lagging strand replication are coordinated by bridging proteins |
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Definition
| Mcm10? Attaches to helicase (Mcm 2-7) and sliding clamp (PCNA) during replication |
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Term
| Topoisomerases are enzymes that can relieve supertwisting |
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Definition
Type I topoisomerases nick one strand.
(tyrosine at active site)
Topoisomerase I uses the energy of the supertwist to religate the nick
Type II topoisomerases make double strand breaks
Type II require ATP and are essential to the cell. Type I enzymes are not essential to the cell.
Type II efficiently separate two interlocked DNA circles.
DNA Topoisomerases break one or two DNA strands and then re-ligate the strand. Topo I work to reduce superhelical tension and use the energy to religate the strand(s) they have broken.
Topoisomerase II helps to separate two interlocked DNA strands. It requires an input of energy from ATP hydrolysis. |
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Term
| Anticancer and antiviral drugs target DNA replication |
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Definition
Chemotherapeutic agents target the biosynthesis of dNTPs. e.g. 5-fluorouracil and 6-mercaptopurine (6-MP).
Target DNA synthesis directly. e.g. Cystosine arabinoside (AraC).
Damage DNA by blocking DNA replication. e.g. Cisplatin and bischloroethylnitosourea (BCNU).
Replication inhibitors are also used as antiviral drugs. The first drug against HIV was azidothymidine (AZT), a thymidine analog that inhibits the specialized DNA polymerase that copies the RNA genome of HIV into DNA after infection |
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Term
| The strand that is copied is called the template strand and the one that is synthesized to complement it is called the newly made or nascent strand. |
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Definition
The two template strands are replicated differently because all synthesis occurs 5’-3’. Leading strand replication occurs by continuous synthesis into the opening fork. Lagging strand replication occurs by the synthesis of short fragments called Okazaki fragments.
The two template strands are replicated differently because all synthesis occurs 5’-3’. Leading strand replication occurs by continuous synthesis into the opening fork. Lagging strand replication occurs by the synthesis of short fragments called Okazaki fragments.
DNA polymerases catalyze the nucleophylic attack of the 3’-OH of a primer on the alpha-phosphate of an incoming deoxynucleoside 5’-triphosphate chosen by complementation to the template. The primer must be annealed, i.e., base paired, to the template. |
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Term
| How can the ends of linear chromosomes be replicated without becoming shorter with each round of replication? |
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Definition
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Term
| Replication bubble is formed by replication fork initiation |
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Definition
The position where DNA helix is first opened to start replication is called Replication origin.
These contain short DNA sequences that attract initiator proteins.
Origins are A-T rich regions so easy to open.
The structure in which both the parental strands have been separated from each other and serve as templates for DNA synthesis is called the Replication bubble
Bacteria have a single origin of replication. The E. coli chromosome is circular with a single origin
It takes E. coli about 40 minutes to duplicate its genome of 4.6 X 106 nucleotide pairs. |
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Term
| Initiation in E. coli is highly regulated |
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Definition
The initiator protein binds first to the origin and is followed by the helicase that opens the DNA.
Binding of initiator protein to the origin is highly controlled.
a) Replication is initiated only when there are sufficient nutrients available.
b) Hemimethylation of newly synthesized DNA |
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Term
| Methylation controls origin firing |
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Definition
Note that the A in the GATC sequence of the origin is methylated. In its hemimethylated state, the origin of replication is bound by an inhibitor protein which blocks access of the origin to the initiator proteins. Eventually (about 20 minutes after replication is initiated), the hemimethylated origins become fully methylated and can start replication.
DNA replication begins at replication origins. In E. coli, the A in GATC sequence in the replication origin gets hemi-methylated after replication and prevents re-replication. |
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Term
Eukaryotic DNA replication
Replication units
ORC binding sites
Origin Recognition Complex
Abf1-binding site |
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Definition
Human chromosome contains a single linear DNA molecule of about 150 million nucleotide pairs.
It is estimated that replication occurs at the rate of 50 nucleotides per second.
At this rate it would take 800 hours to replicate such a DNA molecule from one end to another.
However, eukaryotes have overcome this problem by having multiple origins of replication, activated in clusters called Replication units.
For budding yeast (S. cerevisiae), location of every origin of replication on the chromosome has been determined.
This is chromosome III in budding yeast. It is the one of the smallest eukaryotic chromosomes known. It carries 180 genes. There are 19 origins of replication. Secondary origins are used only 10% of the time while Primary origins are used 90% of the time.
Yeast replication origins have three components.
ORC binding site- a sequence where a protein complex ORC (Origin recognition complex) binds and initiates replication.
Unwinding region – an AT rich region that can be easily unwound.
Abf1-binding site, Binding site for proteins that facilitate ORC binding.
The best known eukaryotic replication origins are ARS elements in yeast chromosomes. They bind a six protein complex called ORC (origin recognition complex). |
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Term
| DNA is replicated once and only once during S phase |
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Definition
During the G1, S and G2 phases, the cell grows continuously.
During M phase growth stops, the nucleus divides, and the cell divides into two.
G1 is the gap between M and S phase; G2 is the gap between S phase and M phase.
DNA replication occurs only in S phase. |
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Term
Replication initiation is regulated
Origin licensing
Cdc6
Cdt1 |
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Definition
Three steps to licensing:
- ORC (origin recognition complex) consists of six subunits. It binds to the origin. It hydrolyzes ATP.
- The hexameric Mcm helicase is loaded by the loaders Cdc6 and Cdt1 to form the prereplication complex (pre-RC)
- Cdc6 is phosphorylated and then degraded by the proteasome. ORC is also phosphorylated. The degradation of Cdc6 and the phosphorylation of ORC prevents origins from firing more than once |
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Term
| Telomerase helps telomere replication |
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Definition
Telomerase is a DNA polymerase of the reverse transcriptase family.
It carries its own RNA template. It elongates DNA in the 5’ to 3’ direction.
A nuclease chews back the 5’ end ensuring a 3’ DNA end that is always longer than the 5’ end (leaves a protruding single stranded DNA).
Telomerase adds telomeric repeats
Telomerase is a large RNA-protein complex. The RNA contains a templating sequence for synthesizing new DNA telomere repeats. The synthesis reaction itself is carried out by the reverse transcriptase domain of the protein. |
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Term
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Definition
| Electron microscopy reported here demonstrated that TRF2 can remodel linear telomeric DNA into large duplex loops (t loops) in vitro. Electron microscopy analysis of psoralen cross-linked telomeric DNA purified from human and mouse cells revealed abundant large t loops with a size distribution consistent with their telomeric origin. Binding of TRF1 and single strand binding protein suggested that t loops are formed by invasion of the 3' telomeric overhang into the duplex telomeric repeat array. T loops may provide a general mechanism for the protection and replication of telomeres. |
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Term
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Definition
| A mutation is a change in our DNA sequence. |
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Term
Silent mutations
synonymous |
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Definition
Because only 1.5% of our DNA comprise codons, changes in the other 98.5% of our DNA sequence are largely inconsequential.
Even within the DNA that encodes amino acid residues of proteins (codons), some changes do not alter the primary sequence of the protein because the code is degenerate. For e.g. mutation of TAA to TAG will still code for a STOP codon.
The term synonymous is used to indicate a mutation that alters the DNA but not the amino acids. |
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Term
Polymorphisms
SNPs (Snips) |
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Definition
The two copies of each of your DNA molecules are not the same. They are somewhere between 98% and 99% the same. Most of the differences are inconsequential and are called polymorphisms. Polymorphism has come to mean recognizable variation between DNA sequences, consequential or inconsequential.
Many of these differences are a single base pair difference and are called SNP (snips) for single nucleotide polymorphism. |
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Term
Mutational changes that alter the amino acid sequence of a protein
Recessive
Dominant |
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Definition
Mutations that alter the behavior of a protein.
Recessive: A mutation that does not manifest a phenotype in the presence of a normal copy. Mutations that destroy the activity of a protein are often tolerated because we have two copies of most genes, and we can get by with half the amount of most proteins.
Dominance: A mutation that manifests itself in the presence of a normal copy. |
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Term
Point mutations: Substitution of one base pair for another.
A point mutation can alter one amino acid |
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Definition
a) Transition: purine for purine; pyrimidine for pyrimidine
b) Transversion: purine for pyrimidine; pyrimidine for purine
Missense mutation - a nucleotide change that results in a different amino acid such as change from glutamate (GAA) to glutamine (CAA).
Nonsense mutation – change in the nucleotide sequence so that instead of coding for an amino acid it now codes for a stop codon.
Silent mutation is used to indicate a mutation that alters the DNA but not the amino acids. |
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Term
| The two-step process of a point mutation |
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Definition
a) Replication error
b) Loss of nucleotide - any replacement |
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Term
| Point mutations cause human diseases |
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Definition
Sickle-cell anemia – resulting from a transversion that produces an amino acid change in hemoglobin, a GAG-encoding glutamate to GTG, encoding Valine.
Werner syndrome – causes premature aging due to genetic instability.
Genes in DNA repair - Cancer |
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Term
Small insertion and deletion mutations change protein length
Indels |
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Definition
Deletions - Insertions: Indels
Indels are usually thought of as deletions or additions of one or a few bases |
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Term
| Insertions and deletions can lead to frameshift mutations |
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Definition
| Frameshift mutation –A frameshift mutation happens when enough bases are inserted or deleted to disrupt the entire triplet coding of amino acids in the DNA template. This is serious as the gene is destroyed and the protein can no longer be made. |
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Term
| Insertions and deletions can lead to frameshift mutations |
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Definition
Some mutations are very large and form abnormal chromosomes
Deletion, Duplication, Inversion
Multiple chromosome mutations (insertions, translocations)
Philidelphia chromosome (22 & 9) |
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Term
| Causes of mutation: 1. The chemical environment of cells |
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Definition
DNA is susceptible to spontaneous changes even under normal cell conditions. If left unrepaired these could lead to mutations.
Depurination releases Guanine and Adenine from DNA.
Deamination converts Cytosine to Uracil.
Modification of nucleotides produces mutations
8-hydroxyguanine (8oxoG) – highly mutagenic compound. It is generated when DNA is attacked by reactive oxygen species (ROS). ROS is a byproduct of oxygen metabolism.
When it pairs with Adenine, it will give rise to G:C to T:A transverison |
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Term
| Causes of mutation: 2. Environmental insults - UV light, X-radiation |
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Definition
UV light can induce covalent linkages between two adjacent pyrimidine bases in DNA to form for e.g. thymine dimers.
X-rays are hazardous because they cause double strand breaks in the DNA which are difficult to repair. They directly attack the deoxyribose in the DNA backbone. They also produce ROS.
X-rays are used to kill rapidly producing cells during cancer treatment. |
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Term
| Causes of mutation: 3. Enzymatic mistakes |
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Definition
| What this says is that every time you replicate your DNA, one error remains! |
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Term
DNA Repair Systems: 1. Mismatch repair
MutS and MutL |
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Definition
Mutant DNA polymerase no longer can proofread, results in many replication errors.
Only on the newly synthesized strand.
It is a three-step method
1. recognition
2. excision of mismatch segment
3. resynthesis of excised segment
Individuals who carry a defective copy of mismatch repair gene are predisposed to certain types of cancer.
Mismatch on template strand - gap in daughter strand - MutS and MutL combine and remove strand from gap to error. |
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