Lecture 3 (1.3)
5'-3'
General DNA polymerase summary, point 3: All DNA polymerases synthesize in this direction.
a template-dependent manner
in vivo, how is DNA synthesized?
A right hand: palm, thumb, and fingers
After what item can polymerase domains be modeled?
Accuracy of the template-cognate dNTP selection
Define "fidelity"
DNA polymerase I 5'-3' exo & polymerase activities can replace DNA at a nick
Define "nick translation"
nucleotides added before polymerase dissociates
Define the processivity of a polymerase.
Yes
Does DNA Polymerase I of E. coli have 3'-->5' Exonuclease activity, aka proofreading?
Yes
Does DNA Polymerase I of E. coli have 5'-->3' Exonuclease activity?
yes
Does DNA Polymerase II of E. coli have 3'-->5' Exonuclease activity, aka proofreading?
no
Does DNA Polymerase II of E. coli have 5'-->3' Exonuclease activity?
yes
Does DNA Polymerase III of E. coli have 3'-->5' Exonuclease activity, aka proofreading?
no
Does DNA Polymerase III of E. coli have 5'-->3' Exonuclease activity?
E.coli DNA polymerase I has 3 distinct activities in 3 distinct protein domains
E. coli DNA polymerase I has how many distinct activities in how many distinct protein domains?
N cycle amplifies the target sequence 2^N -fold
Elaborate on how PCR works
Imagine you are holding a drink in your right hand. The DNA is like a long, continuous string: the string comes in to where your fingers are cupped, and leaves from your fingers, still in a straight line, parallel to your arm such that the string passes under your thumb. Outside of your hand is the template strand only, and the 5' end of that strand. Inside of your hand is the newly synthesized dsDNA, and the 3' end of the template strand and the 3' and 5' ends of the primer strand. An incoming nucleoside triphosphate comes in through the space between your forefinger and thumb, and attaches to and thus elongates the new primer strand. As the primer strand grows, your hand, or the DNA polymerase, moves down in the direction of the 5' end of the template strand, so that the DNA as a whole grows. An exonuclease is found in the space of the outer edge of your palm.
Explain the right hand model in the context of DNA synthesis.
size
Gel electrophoresis separates DNA by _____
DNA polymerases specialized for different cellular roles
General DNA polymerase summary, point 1: Organisms have multiple ______________
template, primer, dNTP, and magnesium. As will be discussed in a later class, some polymerases with DNA repair roles act without needing an intact template, but none of them function without a primer
General DNA polymerase summary, point 2: DNA polymerases generally share 4 requirements for function. What are they?
A 3'-5' exonuclease. Mismatches in the primer-template duplex near the 3' OH disfavor polymerase activity, and favor 3'-5' exo activity
General DNA polymerase summary, point 4: Proof-reading during the DNA synthesis process by what reduces errors?
A 5'-3' exonuclease that converts duplex to single-stranded DNA.
General DNA polymerase summary, point 5: E. coli DNA pol I has a unique additional activity. What is this?
processivity. Some polymerases dissociate after adding just a few dNTPs, e.g., DNA pol I. Others are much more processive, e.g., DNA pol III.
General DNA polymerase summary, point6: Different polymerases have different ___________
1 type of subunit
How many different types of subunits does DNA Polymerase I of E. coli have?
greater than or equal to 4
How many different types of subunits does DNA Polymerase II of E. coli have?
3 in the Pol III core, + other accessory factors
How many different types of subunits does DNA Polymerase III of E. coli have?
7 bp in the minor groove
In the example from class, what does the phage T7 DNA polymerase interact with?
ONLY in the 5'-3' direction
In what direction does DNA synthesis occur?
Yes
Is the polymerase active site mutant viable for Pol I of E. coli?
Yes
Is the polymerase active site mutant viable for Pol II of E. coli?
no
Is the polymerase active site mutant viable for Pol III of E. coli?
mismatch repair, proofreading, nucleotide selectivity
List "nucleotide selectivity," "mismatch repair," and "proofreading" in order of involvement with increasing base substitution rate
A 5'-->3' (forward) exonuclease activity mediating nick translation during DNA repair; a 3'-->5' (reverse) exonuclease activity that mediates proofreading; a 5'-->3' (forward) DNA-dependent DNA polymerase activity, requiring a 3' primer site and a template strand
List the 3 distinct activities of E. coli DNA polymerase I. ???
ethidium bromide; acridine orange
Name two common DNA fluorescent dyes
DNA repair; these specialized DNA repair polymerases will be described in later lectures
Now, there are known to be additional DNA polymerases specialized for what?
Polymerase Chain Reaction
PCR
isolate and amplify and known DNA sequence
PCR can _________________
Grow E. coli bacteria
Step 1 of Arthur Kornberg's biochemical strategy for developing an in vitro DNA polymerase assay
The unpaired 3'-OH end of the primer blocks further elongation of the primer strand by DNA polymerase. The exonuclease starts at the 3' end of a chain, and moves 3'-5'. Note the red primer strand.
Step 1 of the 3'-5' exonuclease "proof-reading" process
The mispaired end is transferred to exonuclease active site, and the mispaired base is excised.
Step 1 of the switching process, if a mismatched base is encountered
Break open the cells
Step 2 of Arthur Kornberg's biochemical strategy for developing an in vitro DNA polymerase assay
The 3'-5' exonuclease activity attached to DNA polymerase chews back to create a base-paired 3'-OH end on the primer strand. The 3'-5' exo polarity is opposite of the polarity of polymerase activity: polymerase activity must HALT to allow 3'-5' exonuclease activity
Step 2 of the 3'-5' exonuclease "proof-reading" process
The end is transferred to the polymerase active site, and DNA synthesis restarts
Step 2 of the switching process, if a mismatched base is encountered
Prepare soluble enzyme extract
Step 3 of Arthur Kornberg's biochemical strategy for developing an in vitro DNA polymerase assay
dNMP is released as a product, leaving a 3' OH. DNA polymerase continues the process of adding nucleotides to the base-paired 3'-OH end of the primer strand.
Step 3 of the 3'-5' exonuclease "proof-reading" process
Fractionate extract to resolve different cellular components from each other; repeat; repeat
Step 4 of Arthur Kornberg's biochemical strategy for developing an in vitro DNA polymerase assay
Search for DNA polymerase activity using a biochemical assay: incorporate radioactive mononucleotides into polynucleotide chains
Step 5 of Arthur Kornberg's biochemical strategy for developing an in vitro DNA polymerase assay
Active sites for synthesis and editing
The 3' end of the growing chain must switch between what?
Polymerase active site discrimination for correct helix geometries; the balance of 5'-3' polymerase and 3'-5' exonuclease activities
The frequency of incorrect nucleotide incorporation, or base substitution rate or error rate, depends on what? 2 items.
False; some wrong additions are not removed, some correct additions are removed
True or false: discrimination in the synthesis/proofreading process is perfect
False; replacing RNA or DNA in a nicked duplex with new DNA has important roles in DNA replication and DNA repair
True or false; replacing RNA or DNA in a nicked duplex with new DNA is not that important.
ds/ssDNA junction with 5' overhang
What "junction" does proof-reading require?
polymerase activities; roles of repair, primer synthesis
What are the activities and role(s) of the Eukaryotic DNA Polymerase alpha?
Polymerase, 3'-->5' exonuclease activities; roles of genome replication (lagging strand) and repair
What are the activities and role(s) of the Eukaryotic DNA Polymerase delta?
Polymerase, 3'-->5' exonuclease activities; roles of genome replication (leading strand) and repair
What are the activities and role(s) of the Eukaryotic DNA Polymerase epsilon?
Polymerase, 3'-->5' exonuclease activities; role of mitochondrial DNA replication
What are the activities and role(s) of the Eukaryotic DNA Polymerase gamma?
single-stranded template; annealed primer with 3' OH; deoxyribonucleotides with 5' triphosphate, or dNTPs; magnesium ions
What are the four requirements of DNA-templated (DNA-dependent) DNA polymerases?
specialized roles in DNA repair
What do additional eukaryotic DNA polymerases have?
cleaves internally
What does "endo" mean in this context?
cleaves from the 5' or 3' end
What does "exo" mean in this context?
Disfavors polymerase activity; favors proofreading activity
What does incorrect nucleotide addition disfavor? Favor?
polymerase "proof-reading"
What does the 3'-5' exonuclease that DNA polymerase I has, provide?
Adds to the DNA chain, while inorganic pyrophosphate (PPi) is released
What does the dNMP (monophosphate) do in DNA synthesis?
Active site "closure" occurs
What happens after dNTP binding?
The DNA polymerase checks the "fit" of base pairing and base stacking before catalysis
What happens in Active site "closure?"
The finger grip tightens to make the active enzyme conformation
What happens in the "closed hand" position?
The green "fingers" and red "thumb" are NOT tight around the DNA, found in the blue "palm" active site
What happens in the "open hand" position?
Aspartic acid side chains that position metal ions to facilitate the primer 3'OH attack on dNTP
What important non-ion things do polymerase active sites have?
genome replication
What is Pol III polymerase activity required for?
an enzyme that disrupts the phosphodiester backbone
What is a nuclease?
an analysis, as of an ore or drug, to determine the presence, absence, or quantity of one ore more components; also, a test used in this analysis
What is an assay?
An enzyme that works by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3' or the 5' end occurs. The exonuclease plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair.
What is an exonuclease?
fluorescence
What is the concept you see in action when ethidium bromide emits visible light?
Mainly functions in the repair of damaged DNA
What is the main purpose of E. coli DNA polymerase I?
polA
What is the name of the structural gene for DNA Polymerase I of E. coli?
polB
What is the name of the structural gene for DNA Polymerase II of E. coli?
polC
What is the name of the structural gene for DNA Polymerase III of E. coli?
16-20 nucleotides/sec
What is the polymerization rate of DNA Polymerase I of E. coli?
n/a
What is the polymerization rate of DNA Polymerase II of E. coli?
250-1000 nucleotides/sec
What is the polymerization rate of DNA Polymerase III of E. coli?
3-200 nucleotides added before polymerase dissociates
What is the processivity of DNA Polymerase I of E. coli?
n/a
What is the processivity of DNA Polymerase II of E. coli?
greater than or equal to 500,000 nucleotides added before polymerase dissociates
What is the processivity of DNA Polymerase III of E. coli?
DNA polymerase interaction with the minor groove
What is the product-template duplex monitored by?
5'-3' polymerase activity
What kind of activity does DNA polymerase carry out?
agarose
What kind of gel is best used in gel electrophoresis for big fragments of DNA - typically <100 bp?
acrylamide
What kind of gel is best used in gel electrophoresis for small fragments of DNA - typically < 500 nt?
Magnesium ions
What kind of ions do polymerase active sites have?
Absorbs UV light; typically, ~254 nm. Emits visible light; orange, ~590 nm.
What kind of light does ethidium bromide absorb? Emit?
5'-3' direction
When DNA is synthesized, nucleotide 5' triphosphate substrates are joined in which direction?
Arthur Kornberg
Who discovered the first DNA polymerase?
in vivo
what is another way to say "in the cell?"