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?"