Term
| Johann Friedrich Miescher |
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Definition
| Discovered DNA 1869. Extracted a viscous substance from isolated white blood cells. Prepared nuclei of cells by adding pepsin. Chemical analysis of this substance found in nuclei ("nuclein") showed that it was 14% N and 2.5% P, different from other known biochemicals. |
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Term
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Definition
Building blocks of DNA, 700 kilodaltons each.
Consists of a 5 C sugar where the 1st C is covalently bonded to a N base, and the 5th C is bonded to a phosphate.
Can be converted to nucleosides by hydrolysis. |
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Term
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Definition
| A N base bonded to an unphosphorylated sugar. Include Adenosine, Guanosine, Cytidine, and Thymidine. |
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Term
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Definition
The 5 C sugar of DNA. The 2nd C is linked to an H atom instead of an OH group.
The OH group on the 3 C is used in forming the phosphodiester bond. |
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Term
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Definition
Planar Carbon-Nitrogen ring structures. The four used in DNA are adenine, guanine, cytosine, and thymine.
N bases with a single ring structure are pyrimidines (thymine, cytosine).
N bases with double ring structures are purines (adenine, guanine). |
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Term
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Definition
Hydrated form of DNA. Standard form. 10.5 steps/base pairs per turn.
Right-handed helix.
Center of symmetry is down the middle of the helix. |
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Term
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Definition
Dehydrated DNA.
11 bases per turn.
Right-handed helix.
Center of symmetry along the outside of the helix. |
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Term
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Definition
Left-handed helix.
12 base pairs per turn.
Has altered geometry of the sugar-base bonds. Can be found in chromosomes where DNA is under torsional stress from unwinding. |
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Term
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Definition
Some nucleotide modifications (e.g., methylation) are found in nature and affect gene function. Chemical modifications of nucleotides (deamination, methylation, additions, etc) change their properties.
Environmental effects (chemicals, radiation) can modify nucleotides and genes, resulting in cancer. |
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Term
| Restriction Modification System |
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Definition
| Bacteria and viruses use modified nucleotides in their DNA so they can tell their DNA apart from invaders. This allows them to target foreign DNA. |
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Term
| 5' to 3' Direction of DNA |
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Definition
The 5' phosphate group of a nucleotide attaches to the 3' hydroxyl group of the last nucleotide on the DNA chain.
DNA is conventionally read in a 5' - 3' direction. The addition of nucleotides in this order gives DNA polarity. |
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Term
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Definition
| Formation of H bonds between two complimentary strands of DNA. Single strands of the same DNA sequence will not hybridize to each other. |
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Term
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Definition
| Determined that the amount of A in DNA matched the amount of T, and the same for G and C. |
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Term
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Definition
| Used equilibrium density centrifugation on a cesium gradient to demonstrate semi-conservative DNA structure. They grew "heavy" DNA in a culture with 15N. After they switched the bacteria to a culture with 14N they could separate the hybrid 14N:15N DNA molecules as the bacteria replicated. These molecules were of intermediate density compared to ones from only 14N or 15N cultures. |
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Term
| Semi-Conservative DNA Replication |
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Definition
| The order of nucleotides in DNA is conserved across generations and throughout cell division because each strand of the parent double helix acts as a template for a newly forming strand. The new, complete DNA molecule has one new strand and one parent strand, so DNA replication is semi-conservative and not dispersive. |
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Term
| Nucleotide Addition to DNA |
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Definition
Once DNA is unwound so that the two strands can act as templates, the new strand is elongated by H bonds forming between the newest nucleotide on the growing chain and its complimentary base on the parent strand.
The 3' OH group attacks the phosphorous atom of the phosphate group on the H bonded nucleotide triphosphate. A phosphodiester bond forms between the last nucleotide added and the new one, and an orthophosphate is released.
Synthesis happens 5'-3' |
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Term
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Definition
| The area of DNA where the helix is unwound and both strands are being replicated simultaneously. Both strands must be read in a 3'-5' direction. The Leading Strand is synthesized in one whole piece from the 3'-5' parent strand. The Lagging Strand is synthesized in short pieces (Okazaki Fragments) from the 5'-3' parent strand. |
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Term
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Definition
| The lagging strand being synthesized from the 5'-3' parent strand is created in short bursts. The replication apparatus jumps 1,000 bases ahead towards the replication fork and then copies backwards. It does this over and over again, creating short fragments of DNA. |
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Term
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Definition
| DNA can only be synthesized if there is a 3' OH already in place for new nucleotides to connect to. To initialize replication, primase enzymes synthesize short (6-11 bp) RNA primers one the parent strand to provide a starting point for DNA synthesis. The lagging strand must continually have new RNA primers added to it for each Okazaki fragment. |
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Term
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Definition
The DNA Replication complex.
Contains all proteins required for DNA unwinding and replication. |
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Term
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Definition
| Protein that unwinds and untangles DNA so that it can be replicated. |
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Term
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Definition
| After DNA has been primed and synthesized, this enzyme hydrolyzes the RNA primer from the template strand, leaving a gap that DNA polymerase will fill. |
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Term
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Definition
| Enzymes that catalyze DNA replication. The first one shown to act in Prokaryotic DNA replication was Pol I. Pol II and Pol III were discovered later. Pol III does the majority of polymerization in bacteria. Pol I and Pol II repair gaps and discontinuities. |
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Term
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Definition
| A multisubunit protein. Polymerase III is a holoenzyme with 10 subunits. 2 subunits function in DNA polymerization, one for the leading strand and one for the lagging strand. |
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Term
| DNA Polymerase Exonuclease Function |
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Definition
| This function of DNA polymerases helps ensure the accurate replication of DNA. Copying errors lead to base changes (mutations) in DNA. The exonuclease fragment can detect base pair mismatches and replace them with the correct base, proofreading the DNA while it is being synthesized. It does this in a 3'-5' and will also replace mismatches in the primer sequence before replication begins. |
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Term
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Definition
These proteins function in polymerizing, pyrophosphorolysis, and pyrophosphate exchange. The last tow are the opposite of polymerization, i.e., they degrade DNA.
Polymerases can polymerize in the 5'-3' and degrade in both the 5'-3' and 3'-5'.
There are two domains to the protein, the small exonuclease unit to degrade, and the larger Klenow fragment to polymerize.
Also hold together sister chromatids to ensure proper recombination. |
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Term
| Conserved Structure of DNA Polymerases |
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Definition
| Two unrelated bacterial groups have the same organization of proteins in their DNA synthesizing holoenzyme, indicating that there are a limited number of organizational options for ensuring polymerization activity. Bacterial polymerases can replicate DNA from a wide range of sources. |
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Term
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Definition
At a nick in one strand of DNA, Pol III can add nucleotides to the 3' end of the nick and also remove them from the 5' end of the strand in front of it. This simultaneous polymerization and hydration continues until the enzyme is dislodged and the nick is closed by ligase.
Used in vitro to include labeled nucleotides into DNA molecules. |
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Term
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Definition
| A DNA polymerase that can create polynucleotide chains sans template. It will continue to add nucleotides to a DNA strand even without base pairing to a template. Used to generate 3' labeled species. |
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Term
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Definition
Polymerases from different sources have different functions and sequence structures and are divided into families: A, B, C, and X.
DNA polymerases from the A and B families are usually used in biotech applications because of their monomeric protein structure. |
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Term
| Chemically Altered Polymerases |
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Definition
Different amino acid structures are designed to produce polymerases with altered functions to use in the lab. Alterations include:
Increased processivity - staying on the template longer to produce longer products
Increased fidelity - faithful template copying
Increased substrate specificity - affinity for altered nucleotides |
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Term
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Definition
Pol I from E. coli does repair, recombination, replication.
T5 Pol, T7 Pol are from T5/T7 bacteriophages and function in replication.
Pol gamma from mitochondria does replication. |
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Term
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Definition
Pol III from E. coli does repair.
Archael from P. furiosus does replication and repair.
Phi29 Pol and T4 Pol from phi29 and T4 bacteriophages do replication.
Pol alpha, Pol delta, and Pol epsilon from eukaryotes do repair.
Viral pols do repair. |
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Term
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Definition
Pol III core from E. coli does replication.
dnaE, DNAEbs from B. subtilis do replication. |
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Term
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Definition
| Pol beta from eukaryotes does repair and replication. |
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Term
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Definition
Pol eta, Pol iota from eukaryotes do bypass replication.
Pol kappa from eukaryotes does bypass replication and cohesion.
Pol IV, Pol V from E. coli do bypass replication.
Rev 1, Rad 30 from S. cerevisiae do bypass replication and UV-induced repair.
Rad 6, Pol xi are from S. cerevisiae. |
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Term
| Eukaryotic DNA Polymerases |
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Definition
The nucleus is home to alpha and beta polymerase protein complexes. Mitochondria contain gamma polymerases.
Have less noticeable exonuclease activity than prokaryotic polymerases.
Polymerase delta, found in bone marrow, had 3'-5' exonuclease activity.
Alpha is the most active and associated with chromosome replication. Beta and delta do DNA repair. |
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Term
| Thymine/Pyrimidine Dimers |
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Definition
| DNA rpair enzymes repair distortions in the DNA duplex caused by aberrantly shaped bases or mismatches using their 5'-3' exonuclease function. This degrades DNA from the 5' end and can also cleave diester bonds several bases from the chain's end. It removes lesions like thymine or pyrimidine dimers, structures caused by UV light where adjacent Cs an Ts become joined. |
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Term
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Definition
| Endonucleases that recognize specific sequences of DNA and break the DNA backbone. These function in prokaryotes as primitive immune agents, destroying foreign bacteria that invades the cell. They recognize cutting sites based on base sequence and methylation. |
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Term
| Type I Restriction Enzymes |
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Definition
| Have both nuclease and methylase capabilities. Their binding sites contain methylated adenines and consist of 4-6 nucleotides separated by 6-8 bp. The site of cleavage can be 1,000 or more bp away from the binding site. |
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Term
| Type III Restriction Enzymes |
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Definition
| Can both methylate and cut DNA. Their recognition sites are asymmetrical and they cleave DNA 24-26 bp in the 3' direction from the binding site. |
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Term
| Type IV Restriction Enzymes |
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Definition
| Can cut and have methyltransferase function. Can also methylate DNA. Cut DNA 10 bp from the binding site. |
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Term
| Type II Restriction Enzymes |
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Definition
| Most commonly used in labs. Don't methylate. Bind to symmetrical DNA sites as dimers. The recognition sites are palindromic on both strands of DNA (bilateral symmetry). Unlike the others, they cleave at the binding site. Not found in eukaryotes. |
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Term
| Type II Restriction Enzyme Double-Stranded Breaks |
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Definition
Some cut with staggered separation at the binding site, leaving overhangs of 2-4 bp that can hybridize with complimentary ends on other DNA fragments (sticky ends).
Some cut the DNA strands in the same place, leaving flush (blunt) ends.These cna also be joined, just less efficiently. |
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Term
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Definition
| Blunt ends of restriction fragments can be joined to any other fragment with blunt ends, whether the fragments were cut by the same enzymes or not. Sticky ends can only be joined if they have matching overhangs. Sticky ends can be converted to blunt ends using DNA polymerase to extend the recessed end, or by using exonucleases to remove the overhanging bases. Synthetic DNA fragments can be used as adapters if they have one blunt end and one sticky end. |
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Term
| Unusual Type II Restriction Enzymes |
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Definition
Type IIs (Fok1, Alw1) recognize 5-7 non-palindromic sequences, cut DNA within 20 bases of the binding site.
Type IIm (BisII, DpnI) cut methylated DNA. Useful for detecting DNA methylation. |
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Term
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Definition
| Catalyzes the formation of phosphodiester bonds between 3' OH and 5' P ends of nucleotides. Functions in the completion of the lagging strand's DNA backbone, strand exchange, or repair synthesis. Can join separate DNA strands. |
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Term
| RNA Ligase vs. DNA Ligase |
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Definition
| Both join DNA or RNA ends. DNA ligase is better at joining DNA ends. In bacteria, DNA ligase converts open or nicked DNA circles to closed circles, protects free DNA ends, and extends DNA onto overhanging templates. It can also recover transformation-capable DNA after nicking. |
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Term
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Definition
Unlike endonucleases (which break phosphodiester bonds), exonucleases degrade DNA at free 3' OH or 5' P ends. They can't work on closed DNA circles.
Can be used to make stepwise deletions in linear DNA or modify cut ends of DNA from restriction digests.
Have specific substrate requirements. |
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Term
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Definition
| An exonuclease from E. coli. Degrades ssDNA from the 3' end into mononucleotides. Works best on long, single-stranded ends, less well on double-stranded regions. |
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Term
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Definition
From E. coli. Works in the presence of Mg2+ and Mn2+ to remove 5' nucleotides from 3' ends of dsDNA.Can also work as an endonuclease at apurinic sites on DNA (sites without a purine, maybe a mutation or damage). Can remove nucleotides from blunt or recessed ends and nicks, but not from 3' overhangs.
Used to create nested deletions in dsDNA or to produce ssDNA for dideoxy sequencing. |
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Term
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Definition
| From E. coli. One of only a few enzymes with 5' exonuclease activity. Can degrade ssDNA from 3' or 5' end. Can remove long single strands protruding from dsDNA. |
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Term
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Definition
| A nuclease from Altermonas espenjiani which degrades dsDNA and ssDNA from either end. Useful for making nested deletions because of its slow activity at 20 C. |
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Definition
| From mung bean sprouts. Digests ssDNA and RNA. Used to remove overhangs on restriction fragments and leave blunt ends because it doesn't touch dsDNA. |
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Definition
From Aspergillus oryzene, Hydrolyzes ssDNA and RNA into 5' nucleotides. Can act as an endonuclease on ss regions of DNA such as gaps or loops.
Used for early RNase protection assays of gene expression. Also used for nuclease mapping techniques. |
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Term
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Definition
| From E. coli. Dependent on ATP. Works on ssDNA and dsDNA. No activity at nicks, but can digest ssDNA at the 3' or 5' end. Can create short fragments (oligonucleotides) from dsDNA. |
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Term
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Definition
Digests ssDNA, dsDNA, and RNA at AT or AU rich sites. Prefers single-stranded substrates.
Used to remove nucleic acid from crude extracts and to analyze chromatin structure. |
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Term
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Definition
| Deoxyribonuclease I. From bovine pancreas. Digests both ssDNA and dsDNA at pyrimidines into oligodeoxyribonucleotides (so technically an endonuclease). Used to remove DNA from RNA extractions. |
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Term
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Definition
| Cellular lysates naturally contain exo and endonucleases, which must be inactivated when preparing nucleic acids for analysis. DNA extraction procedures are designed to minimize their activity. TE buffer chelates cations required for nuclease activity. |
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Term
| Helicase Nicking Activity |
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Definition
| Helicases can break both DNA strand backbones (double-stranded break) or just one (single-stranded break or nick). With a nick, the broken ends can rotate around the unbroken strand. The ends can be digested by exonucleases or extended using the intact template strand (nick translation). |
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Term
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Definition
| To relax DNA from a tightly coiled and looped form so it can be read, helicase enzymes untangle DNA by cutting and reclosing the phosphate-sugar backbone. This relieves topological stress in super-twisted DNA to allow for recombination or transcription. |
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Term
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Definition
Helicases that relax super-twisted DNA or interconvert topological isomers.
In E. coli, Topo I nicks one strand of the double helix to relax supercoils. |
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Term
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Definition
Type II topoisomerases. Cause double-stranded breaks to untangle DNA. Can also separate concatamers (linked rings of DNA).
In E. coli, Topo II (gyrase) can also cause coiling by cutting both strands and passing one part of the duplex through before re-ligating. |
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Term
| Topoisomerases as Cancer Drug Targets |
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Definition
| Anticancer drugs such as camptothecin, epipodophyllotoxins VP-16 and VM-26, amsacrine, intercalating anthracycline dericatives doxorubicon and motoxantrone target toposiomerases. By inhibiting them, this drugs case cell death by preventing breaking and joining activities, possibly leaving broken and unfinished intermediates. |
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Term
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Definition
In prokaryotes: most DNA is methylated or at least hemimethylated to tell host DNA apart from invading DNA, providing resistance to restriction enzymes.
In eukaryotes: DNA is methylated in certain regions. DNA binding proteins may limit methyltransferase access to some regions and guide them to other regions. |
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Term
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Definition
Proteins that catalyze the addition of methyl groups to N bases (usually As or Cs).
Eukaryotic cells have two main tpyes:
Maintenance methyltransferases hemimethylate DNA throughout the whole life of a cell.
de novo methyltransferases work during embryonic development only ad might be responsible for methylated patterns in differentiated cell. |
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Term
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Definition
| The system that provides a predetermined pattern of gene expression during development. Uses methyltransferases such as DmnTI (which works on hemimethylated DNA) and cytosine methyltransferases. Methylated marks DNA for recognition by or resistance to DNA binding proteins in eukaryotes. |
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Term
| Recombinant DNA Technology |
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Definition
| Controlled gene mixing. New genomes are created with single genes altered, replaced, or deleted. This creates organisms like purebreds except for that one gene. |
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Term
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Definition
| Hybrid vigor. A mixed/hybrid individual that will be more robust and well-adapted than a purebred individual. |
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Term
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Definition
| In meiosis, chromosomes duplicate and line up. Chromosomes can cross over or break and rejoin to recombine, creating new duplexes with genes from each chromosome. Recombined chromosomes separate and go to separate gametes. During fertilization gametes from both parents (with recombined chromosomes) join and create offspring with new sets of genes. |
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Term
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Definition
| Asexual recombination between F+ and F- bacteria. The two cells must be in contact so a filamentous bridge can form. Genetic info moves from the F+ to the F- cell but never vice versa. |
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Term
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Definition
Contained in F+ bacteria, carries genetic info to the F- bacteria in addition to forming the filamentous bridge. After the FF is transferred, F- becomes and F+ bacteria. The FF is an extrachromosomal DNA ring that is simultaneously replicated and transferred so that both cells have a copy.
The FF can be lost during cell division, turning a F+ into a F-. |
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Term
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Definition
| Bacteria with chromosomally incorporated F factors (high frequency of recombination). During a crossover or recombination event the F factor can be inserted into the host chromosome. It can still direct mating and carry part or all of the host chromosome across the mating bridge. When that happens another recombination event can insert that info into the host chromosome, forming recombinant genes. |
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Term
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Definition
| Transmission of DNA from one cell to another via viruses such as bacteriophages. This can be used in studies to inject genetic info into strains of bacteria. It can also be used to determine gene order. |
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Term
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Definition
| DNA can be transferred from one cell to another without viruses or a bridge. Broken chromosomal DNA from dead cells can transfer to a living population, but it is an inefficient process. Unprotected DNA can be sheared or degraded. Natural transformation is more effective because the transferred DNA is protected, in a circle or otherwise. |
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Term
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Definition
| ds circles of DNA 2,000-100,000 bp in length. These exist in bacteria outside of the chromosome and carry a limited amount of genetic info. They are supercoiled, requiring nicking to relax the supercoil and breaking to linearize. The different physical states of the plasmid can be detected by gel electrophoresis. |
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Term
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Definition
Plasmids that carry genes for drug resistance. Acridine dyes can kill drug resistance in bacteria, since these dyes induce the loss of episomes (plasmids).
Colicinogenic factors transfer resistance to bacterial toxin proteins (bacteriorins).
Drug-resistant genes can be easily lost or recombined to form new genes. |
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Term
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Definition
Plasmids containing the F factor and possible R factors. They are self-transmissable and contain genes for their own transfer and self-replication.
Occur in small numbers in cells, one or two per chromosome equivalent. |
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Term
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Definition
Numerous in numbers in a bacterial cell, but don't carry genes for self-maintenance. By existing in large numbers they maximize their chance to get taken into cells at cell division or transformation.
Plasmids are a better way to transfer genes into new cells than DNA fragments, since they're more protected. |
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Term
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Definition
Numerous in numbers in a bacterial cell, but don't carry genes for self-maintenance. By existing in large numbers they maximize their chance to get taken into cells at cell division or transformation.
Plasmids are a better way to transfer genes into new cells than DNA fragments, since they're more protected. |
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Term
| Eukaryotic DNA Polymerase Alpha |
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Definition
| Also RNA primase. Forms a complex with a small catalytic (PriS) and large noncatalytic (PriL) subunits. The Pri subunits act as primase. DNA Pol alpha elongates that primer and DNA pol epsilon takes over after 20 nucleotides on the leading strand and DNA pol delta on the lagging strand. |
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Term
| Eukaryotic DNA Polymerase Beta |
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Definition
| Repairs DNA through base excision repair and gap-filling synthesis. |
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Term
| Eukaryotic DNA Polymerase Gamma |
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Definition
Replicates and repairs mintochondrial DNA.
Proofreading 3'-5' exonuclease activity. |
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Term
| Eukaryotic DNA Polymerase Delta |
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Definition
Very processive, proofreading exonuclease activity in the 3'-5'.
Main polymerase in lagging strand synthesis. |
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Term
| Eukaryotic DNA Polymerase Epsilon |
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Definition
Very processive, proofreads in the 3'-5' with exonuclease activity.
Related to DNA Pol Delta and main polymerase in leading strand synthesis, after DNA Pol alpha starts the process. |
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Term
| Prokaryotic DNA Polymerase I |
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Definition
DNA Repair.
Polymerases in the 5'-3'.
Proofreads with exonuclease activity 3'-5'.
Removes primers with exonuclease activity 5'-3'. |
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Term
| Prokaryotic DNA Polymerase II |
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Definition
Repairs DNA.
3'-5' exonuclease activity. |
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Term
| Prokaryotic DNA Polymerase III |
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Definition
Main DNA elongation polymerase.
Proofreads with exonuclease activity 3'-5' |
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Term
| Prokaryotic DNA Polymerases IV and V |
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Definition
| Y-family DNA polymerases. IV acts in bypassing DNA damage. |
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Term
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Definition
Histones are primarily made of lysine and arginine.
Histones form an ionic bond with DNA. |
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Term
| Topoisomerase vs. Helicase |
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Definition
| Helicase separates DNA ahead of the replication fork, topoisomerase unwinds it. |
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