Molecular Bio: Exam 4

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DNA polymerase I (E. coli): what reactions does it catalyze? (685): 1. DNA polymerase activity
2. 3' to 5' exonuclease activity: proofreading
3. 5' to 3' exonuclease activity: DNA degradation of strand ahead of the advancing polymerase
DNA polymerase I (E. coli): what is its general function? (685): important for DNA repair and replacement of initial RNA primer: it can remove and replace a strand all in one pass
process of removing primers and joining nascent DNA frags (fig. 20.29): 1. DNA pol I binds to ds DNA at the nick.
2. pol simultaneously removes the RNA primer and fills in the resulting gap by enxtending the DNA frag in the 5' to 3' direction, leaving degraded primer in its wake.
3. DNA ligase seals the remaining nick with a phosphodiester bond between the left-hand and right-hand DNA frags.
nick translation (685): performed by DNA pol I. Requires the whole molecule because it depends on 5' to 3' degradation of DNA ahead of the moving fork
Klenow fragment: function and structure (685): the large fragment of DNA pol I after proteolysis

Contains polymerase and 3' to 5' exonuclease activity

Structure: contains an O-helix (fingers)
I-helix (thumb)
cleft inbetween (palm): binding site for DNA. Contains aspartate residues coordinated with magnesium, which catalyze polymerase activity
what is the Klenow fragment used for in the lab? (685): can be used for DNA end-filling and sequencing
Taq polymerase structure vs. DNA pol I structure (687): Taq pol has the O, I and palm domains that pol I has
chromatography experiment showing the activity of the different E. coli polymerases (fig. 20.32): METHOD
1. Separated DNA pol from mutant polA1 and wild type E. coli via phosphocellulose chromatography.
2. Since pol III is hidden by pol I, they added N-ethylmaleimide, which inhibits pol III, but not pol I. Thus, pol III activity is measured as the difference between the polI and N-ethylmaleimide curves.

RESULTS
PolA1 mutants showed no Pol I activity, showing that polAI codes for DNA pol I.
genetic experiment showing the activity of different E. coli DNA polymerases (table 20.2): METHOD
1. isolated 15 strains that were temperature-sensitive for DNA replication (in a background of polI mutants)
2. assayed for pol II and III activity

RESULTS
saw that the pol III activity was very temperature sensitive for the dnaE mutants (less activity), but there was no effect on pol II.

CONCLUSION
dnaE makes something contributing to pol III activity and that pol III is necessary for DNA replication
main function of E. coli DNA pol III (688): replication of DNA

the holoenzyme carries out the elongation of primers to make both the leading AND lagging strands of DNA.
which subunits make up the core of E. coli DNA pol III and what are their individual functions? (688): alpha: has DNA pol activity and is the principal subunit.

epsilon: has 3' to 5' exonuclease activity

theta: mostly unknown; stimulates epsilon exonuclease
steps in purification of the alpha subunit of E. coli DNA pol III(fig 20.33): METHOD
1. partially purify DNA pol III from cells that overexpressed the alpha subunit
2. subjected this to purification by ion exchange chromatography and do SDS-PAGE from the peaks.
experiment showing that the alpha subunit of E. coli DNA pol III has polymerase activity (fig 20.33): METHOD
1. pool alpha subunit fractions and purify
2. test for DNA pol activity

RESULTS
Found that the alpha subunit's activity was comparable to the activity of the core polymerase

CONCLUSION
The alpha subunit contributes the DNA polymerase activity to the core.
purification of epsilon subunit of E. coli DNA pol III (fig 20.34): METHOD
1. Used a plasmid with a gene that encodes the epsilon subunit. Put it under control of lambda phage PL promoter (which is heat inducible because the lambda repressor is incorporated at high temperatures, which leads to increased expression).
2. made extracts of uninduced and induced cells and subjected them to SDS-PAGE.
experiment showing the activity of the epsilon subunit of E. coli DNA pol III (20.35): METHOD
1. incubated purified epsilon subunit with 3H-labeled synthetic DNAs and either matched or mis-matched DNA pairs
2. measured the amount of radioactivity remaining the DNAs after increasing lengths of time for the core and epsilon subunit

RESULTS
Found that epsilon had similar activity to the core for excising mismatched base pairs . The radioactivity of the properly paired DNA did not change because there was nothing to excise.
mutator mutants (690): mutants in the dnaQ gene, which encodes the epsilon subunit of E. coli DNA pol III. These mutants are subject to excess mutations (10^3 to 10^5 more so than in wild type) because they do not contain 3' to 5' exonuclease (proofreading) activity.
what is the error rate of DNA replication? (691): 1 mistake per 10^11 base pairs

10^-5 error in the DNA pol III core (1st pass) and the same error in the second pass (3' to 5' exonuclease activity)
why make primers out of RNA? (691): this guarantees that the primers (which have more errors than DNA because they are not proofread) will be recognized, removed, and replaced with DNA by extending the neighboring Okazaki fragment.
eukaryotic DNA pol alpha (691): low processivity

synthesizes primers for both leading and lagging strands
processivity (691): the tendency of a polymerase to stick with the replicating job once it starts
eukaryotic DNA pol delta (691): highly processive

responsible for elongation of both leading and lagging strands

along with PCNA (proliferating cell nuclear antigen), the processivity of delta is enhanced by 40-fold
proliferating cell nuclear antigen (PCNA) and how it works (691): a protein which is enriched in proliferating cells that are actively replicating their DNA. It physically clamps polymerase delta onto the template and allows it to travel 40 times farther without falling off.
eukaryotic DNA pol beta (691): a repair enzyme that is not processive at all

adds 1 nucleotide to chain and dissociates

fills in gaps created when primers/mismatched bases are excised
eukaryotic DNA pol epsilon and pol gamma (691): epsilon: functions in DNA repair

gamma: responsible for replicating mitochondrial DNA
experiment measuring the rate of DNA replication (fig 21.2) second experiment showing processivity of DNA pol III holoenzyme: METHOD
1. constructed a synthetic circular template and labeled the negative strand. Added it to in vitro reactions with pol III holoenzyme plus preprimosomal proteins or dnaB.
2. Took samples from reactions in 10-second intervals, electrophoresed

RESULTS
Found that the rate of replication was 730 nt/sec (close to the in vivo rate of 1000 nt/sec)

2nd experiment was the same except they added competing DNA [poly(dA)] and an antibody directed against the beta subunit of the holoenzyme. Found that it made no difference in rate of replication, meaning that the polymerase is highly processive.
mechanism of rolling circle replication (fig 20.14): 1. an endonuclease creates a nick in the outer (+) strand
2. the free 3'-end created by the nick is the primer for positive strand elongation, as the other end of the positive strand is displaced (negative strand rolls CCW).
3. the unit length of positive DNA that is displaced is cleaved by an endonuclease
4. replication occurs many times until around 20 copies are made
dnaG: function dnaB: function (713): dnaG: a primase made by the dnaG gene in E. coli (other phages like M13 use host RNA polymerase as a primase)

dnaB: a product of the dnaB gene. Facilitates binding of dnaG. Also has helicase activity.
primosome: what is it? How is it different than a primase? (L2): primosome: the collection of proteins needed to make primers for a given replicating DNA (dnaB and dnaG).

A primase primes DNA synthesis at one spot (origin of replication) and a primosome is mobile and can repeatedly synthesize primers as it moves around DNA. A primosome primes Okazaki fragments.
method to locate the E. coli origin of replication (oriC) (fig. 21.3 and 21.4): METHOD
1. clone DNA frags (hopefully including origin) into a plasmid that lacks origin, but has a drug-resistance marker (Amp+).
2. transform into E. coli and select for Amp resistance. Colonies that grow will have both the ori and the Amp+ gene.
3. trim and mutate the DNA frag with the oriC to find the minimal effective DNA sequence.
tell me about the E. coli oriC (fig. 21.5): The consensus sequence is 245 bp long and contains (4) ninemers (dnaA boxes) that are binding sites for dnaA.
1. dnaA binds and cooperates with RNA polymerase and HU protein to melt (3) 13-mer repeats left of the dnaA box. This forms an open complex. RNA pol synthesizes a short piece of RNA, which creates a small loop. HU protein induces bending of dsDNA.
2. DnaB then binds to the single stranded region (open complex), after being brought to the DNA by dnaC.
3. DnaB allows dnaG (primase) to bind, which completes the primosome.
primosome: 2 functions (716): 1. it operates repeatedly in priming Okazaki fragment synthesis to build the lagging strand.
2. dnaB serves as the helicase that unwinds DNA to provide templates for both the lagging and leading strands.
priming in SV40 virus: in which direction(s) does replication occur? where is the origin of replication?(716): Replication occurs bidirectionally from the ori.

The ori overlaps the SV40 central region. (Determined by EM data, cut with 2 different REs.)
5 components of the SV40 ori (fig. 21.8): 1. large T antigen binding sitem made up of 4 pentamers
2. 15-bp palindrome, which is the first region melted during replication
3. 17-bp region of AT pairs, which probably helps melt the palindrome

4. 2 additional large T antigen-binding sites
5. GC boxes to the left of the ori core (bind Sp1)
summary of initiation at SV40 ori (717): 1. large T antigen binds at the SV40 ori and its DNA helicase activity unwinds the DNA
2. primase (from eukaryotic host, associated with DNA pol alpha) synthesizes a primer
experiment to identify the yeast origin of replication (718): METHOD
1. used a plasmid with a putative ARS and isolated replication intermediates
2. linearized rep intermediates with REs
3. did 2D gel electrophoresis: 1st dimension oriented horizontally under low voltage and low agarose concentration, to separate according to size; 2nd dimension vertically under high voltage and high agarose concentration to separate according to shape.
4. Southern blot, probing with labeled plasmid DNA.
behaviors of replication intermediates in yeast (fig. 21.9): 1. simple: ARS is at the extreme end of fragment (y-shaped). Causes an arch on the gel

2. bubble: ARS is in the middle of the frag (bubble). Causes a 1/2 arch on the gel.

3. double Y: a replication bubble split in 2 with part of the bubble on both ends. Causes a linear curve on the gel.

4. asymmetric ARS: bubble converts to a y-shape as it goes along. Causes a discontinuous arch.
experiment that locates the origin of replication in ARS1 (looking at replication intermediates) (21.10): METHOD
Cleave a plasmid with BglII and then PvuI and observe behavior of replication intermediates on the gel.

RESULTS
There is only a single origin of replication in ARS1 or else the pattern on the gel would have been much more complex. With BglII, the pattern is representative of the double Y, meaning that the ARS1 is adjacent to the BglII site.

With PvuI, the gel represents an asymmetric bubble shape, meaning that ARS1 is halfway around the plasmid from the origin.
experiment showing important regions of ARS1 (fig 21.11): METHOD
used linker-scanning mutagenesis

RESULTS
Found 4 important regions: A (most important), B1, B2, and B3 (B3 increased electrophoretic mobility, indicating that it is involved in bending the DNA)
transposons: general definition (L3): DNA that can move (with the help of proteins) from 1 site to another.
replicative vs. nonreplicative transposition (777): replicative: when a transposon inserts itself into a new site and also replicates itself. (eg, Tn3)

nonreplicative: both DNA strands move without replicating
structure of Tn3 transposon (fig 23.5): contains inverted repeats on either end, a tnpA gene (transposase), tnpR gene (resolvase--breaks the cointegrate down into 2 independent plasmids), and a bla gene (which encodes beta-lactamase, which protects bacteria against ampicillin)
2 basic steps of Tn3 transposition (fig 23.6): 1. formation of a cointegrate, in which plasmids are coupled through a pair of Tn3 copies, catalyzed by tnpA.
2. resolution of cointegrate: breaks down into 2 independent plasmids (after crossing over at the 2 res sites), catalyzed by TnpR.
genomic applications of transposons: insertional mutagenesis (L3): insertional mutagenesis: generates gene disruption. Stop codons in every reading frame will disrupt the sequence of the gene wherever it goes. Must include a selectable marker.
genomic applications of transposons: single-gene trap (L3): single-gene trap: the construct has a splice acceptor site in front of a reporter gene (that lacks a start codon and a promoter). The gene trap can only be expressed if the transposon inserts into the exon or intron of a gene. Once splicing occurs, a fusion protein is generated. Gene trapping causes loss-of-function mutations.

Used to identify coding sequences within genomic DNA.

Example: used in the yeast genome, which is exon-rich.
genomic applications of transposons: activation tagging (L4): modifying the transposon to carry a strong promoter or enhancer, which results in overexpression of the host gene.

-a gain of function mutation
-need a selectivity marker

Used to identify genes resulting in a mutant phenotype when overexpressed or expressed abnormally (like redundant or nonessential genes).
genomic applications of transposons: enhancer tags (L4): carries reporter gene with a minimal promoter that can't activate expression on its own. Allows expression only if an enhancer is nearby.

this is a way to identify genes and study expression patterns and a way to select for insertions near genes (usually in large genomes with lots of non-coding sequences)
advantage of using transposons disadvantage of using transposons(L4): advantange: can make a large number of mutants

disadvantage: insertion is not really random, which means that sometimes, you don't get full coverage of the genome.
retrovirus replication cycle (fig. 23.19): 1. reverse transcriptase makes a linear, double-stranded DNA copy of the RNA
2. this DNA copy integrates into the host DNA
3. the host RNA pol II transcribes the provirus, forming genomic RNA.
4. viral RNA is packaged into a virus particle, which buds out of the cell and infects another cell, starting the cycle over again
experiment showing the effect of RNase on reverse transcriptase activity (fig 23.22): METHOD
incubated retrovirus particles with all 4 dNTPs including labeled dTTP under various conditions, and acid-precipitated the product, and measured the radioactivity.

RESULTS
Found that incorporation of the labeled nucleotide was inhibited by including RNase in the reaction, especially incubating the reaction with it beforehand. This showed that RNA was the template in the reverse transcriptase reaction.
retrotransposons: general definition (792): transposons that replicate through an RNA intermediate and therefore depend on reverse transcriptase.
2 types of retrotransposons (792): 1. LTR (long terminal repeat)-containing: replicate like retroviruses (eg. Copia in flies, TY1 in yeast)

2. non-LTR retrotransposons: highly abundant (eg. LINEs). There are 100,000+ in the human genome (17% of genome)
structure of non-LTR retrotransposons (fig 23.28): 1. a 5' and 3' UTR (untranslated region)
2. ORF1: encodes RNA binding protein (p40)
3. ORF2: encodes a protein with an endonuclease and reverse transcriptase activity. Also has a cysteine-rich region.
4. flanked on either side by direct repeats of ORF host DNA.
4. poly-A tail
what do non-LTR retrotransposons use as a primer? (794): The endonuclease (encoded in ORF2) creates a ss break in the target DNA and the reverse transcriptase uses the newly-formed DNA 3' end as a primer.
R2Bm (non-LTR retrotransposon) (794): a LINE-like element from the silkworm. It encodes a reverse transcriptase, but unlike LINES, it has a specific target site--the 28s rRNA gene of the host. It can only insert once in the whole genome.

Since it has this specific target site, it makes the insertion mechanism much easier to investigate.
experiment studying endonuclease and RNA cofactor activity in R2Bm (fig. 23.29): METHOD
1. purified the endonuclease from R2Bm
2. added the purified endonuclease +/- RNA cofactor to a supercoiled plasmid bearing the target site. Electrophoresed.

RESULTS
Found that the ss break in the target DNA occurs 1st and then cleavage of the other strand occurs much more slowly. They knew this because if supercoiled plasmid is nicked once, it forms a relaxed circle (migrates more slowly than supercoiled or linear DNA). If it is nicked twice, it will take the linear form.

When they removed the RNA cofactor, they found that the endonuclease by itself caused rapid ss nicking, but barely detectable cleavage of the other strand.

CONCLUSIONS
RNA (cofactor?) is important in causing the break in the 2nd strand of DNA.
experiment showing the nicked strand of DNA (R2Bm) serves as a primer(fig. 23.30): METHOD
1. Took prenicked target DNA (primer) with the target site close to the left end and added R2Bm RNA (as template) + the ORF2 reverse transcriptase + all 4 dNTPs, including a labeled dNTP.
2. Electrophoresed and autoradiographed products. They expected an 802-nt product, based on the location of the nick.

RESULTS
No product with nonspecific RNA template. A strong 1.9kb band appeared with R2Bm RNA, from the 1kb primer + ~800nt reverse transcript. See other results in figure.

To confirm that the target DNA acted as a primer, they performed PCR with primers specific to the target DNA and reverse transcript and ontained PCR products of the expected size and sequence.
model for L1 transposition (fig. 23.31): 1. the L1 element is transcribed and process.
2. transcript is exported from the nucleus
3. mRNA transcript is translated to yield the ORF1 product (p40) and the ORF2 product. These products associate with the mRNA.
4. the ribonucleoprotein reenters the nucleus.
5. the endonuclease nicks the target DNA (anywhere) and the RT uses the new DNA 3' end as a primer for the reverse transcript.
6. Eventually, the second L1 strand is made and the whole element is liagated into the target DNA.

DRAW
exon shuffling: rearranging exons during transposition
DNA microarray 2 types (830): high-density array of DNA for parallel hybridization analysis

Types
1. spotted DNA microarray: mechanical spotting. Place a very small quantity (nL to pL) of DNA on a microscope slide--> enough for all the genes in a genome

2. oligonucleotide chip (or Affymetrix, or DNA microchip): for oligonucleotides (20-25-mers). Synthesized on a silicon chip.
method for identifying differential gene expression under a given condition (831): Sample 1: extract RNA from normal tissue, generate cDNA labeled with fluorescent Cy3 (green).

Sample 2: extract RNA from tumor cell, generate cDNA labeled with fluorescent Cy5 (red).

Mix the 2 together, hybridize them to the array, quantify the resulting fluorescence.

If a spot is green, it means that the DNA was expressed the most in the normal tissue and less in the tumor cell.

If a spot is red, it means more DNA is expressed in the tumor cell than in normal tissue.

If the spot is yellow, it means DNA was expressed equally in both.

This method allows researchers to see which genes are being transcribed in a cell under a given condition.
growing oligonucleotides on a glass substrate (fig. 24.17: 1. coat glass with a reactive group that is blocked with a photosensitive agent. Mask parts of the plate that you don't want to add a nucleotide to.
2. Expose glass to light; the light will expose the unmasked spots.
3. A blocked nucleotide is chemically coupled to the unblocked spots on the glass.
4. In the next cycle, this nucleotide can be masked or not.
2 functions of microarrays (2 things that can be varied) (L5): In arrays, you can vary:
1. labeled target (eg, extract RNA under different conditions)

2. probes on array (eg, can probe for coding sequences, promoter regions, or both)
regulon (837): a set of genes that tend to be regulated together (eg. when an activator has many enhancer targets in a genome and thus, activates many genes at once)
ChIP-chip procedure for DNA-protein analysis (837): Melding of chromatin immunprecipitation and DNA microarray hybridization

METHOD
1. proteins are chemically cross-linked to DNA (with formeldahyde) in yeast cells (wild type cells and cells missing a gene of interest)
2. cross-linked DNA is extracted and sheared by sonication into 2-3kb frags
3. sheared chromatin is immunoprecipitated with an antibody directed against an epitope tag on the protein of interest.
4. Reverse the cross-links and amplify/labeled precipitated DNA by PCR. Label precipitated DNA with one color and non-precipitated DNA with another.
5. Hybridize DNA to a microarray

RESULTS
If a DNA spot on the microarray hybridizes to DNA that binds to the protein of interest more than to other proteins, that spot will fluoresce a certain color. If the DNA hybridizes to (non-precipitated) DNA that binds other proteins preferentially, the spot will fluoresce the other color. If the spot binds both equally, it will fluoresce an intermediate color.

In vivo technique because you are using chromatin and not a specific protein.
DIP-chip method (L5): DIP-chip = DNA immunoprecipitation with microarray detection

Instead of chromatin, use DNA fragments and one protein of interest

This in vitro technique is useful for comparision with in vivo (ChIP-chip) results.

Allows one to infer chromatin repression of promoters (nucleosome occupancy) (?).
using mass spec to detect protein-protein interactions (fig. 24.26): METHOD
1. generate a tagged bait protein, which is engineered to include the coding region for an epitope tag and then placed in yeast cells and expressed to yield the tagged protein.
2. Use immunoaffinity chromatography with an antibody directed against the epitope tag to purify the protein complex (bait protein + other proteins it interacts with).
3. separate proteins by SDS-PAGE.
4. cut out protein bands from gel and analyze the peptides by mass spec
5. identify components of the protein complex by comparing the masses of the peptides with masses of proteins encoded in the yeast genome.
mass spec, very generally (L5): a very precise way to determine the mass of a peptide and thus, determine its sequence
define protein microarray what are some obstacles to creating them? (L5): protein microarray: proteins are spotted on an array instead of DNA

obstacles: requires purification of a large number of proteins (labor intensive), which all must be attatched to a solid surface while still maintaining their functions.

rare
using a protein microchip to detect protein-protein and protein-lipid interactions (fig. 24.28): METHOD
1. made protein arrays and probed them with an anti-GST antibody or specific probes. The anti-GST antibody was detected with a fluorescent probe to yield a red color. The other probes were coupled to biotin, which could be detected with streptavidin coupled to a green fluorescent tag.

RESULTS
Each green spot corresponds to a protein on the microarray that binds to the protein or lipid probe. The proteins probed with the anti-GST antibody are controls that tell how much of each protein was spotted on the array.

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