Unit 1: DNA

DNA

DNA

deoxyribonucleic acid, molecule that encodes inherited genetic information

nucleotide

building block; each comprised on a sugar-phosphate backbone (of pentose and sugar) and one of 4 bases

what is DNA’s overall charge?

the phosphate group having a negative charge gives the overall DNA a negative charge as well

why do bases have their complimentary pair?

because that pairing (A+T or G+C) is the most energetically favorable

what carbon are the base pairs attached to?

carbon 1

adenine and thymine H bonds

forf 2 H bonds

cytosine and guanine H Bonds

form 3 H bonds

antiparallel strands

because of the way the H bonds form, it forces the sugar-phosphate backbone to go in opposite directions: 5’ to 3’ and 3’ to 5’

functional consequence of this base pairing

each strand of DNA contains a sequence of nucleotides exactly complimentary to the opposite strand. this allows one strand to act as a template to make a copy of the other strand during DNA replication

semi-conservative replication

each daughter DNA molecular contains one strand from the parent DNA molecular and one newly synthesized strand

chromosomes

• separated into manageable units that are organized spatially

• organized subunits of the genome

• each 23 occupies its own space in the nucleus

• (think organized and rolled spools of yarn)

nucleosomes and chromatin

wound tightly to maximize space, leaving specific parts available for use

genome

the complete sequence of DNA (genetic information) present in a cell or organism

gene

unit of genetic information

what does each chromosome equal?

1 molecule of DNA

diploid

2 copies of every chromosome: 1 from biological mother and 1 from biological father

chromatin

• fibers made up of packed nucleosomes

• half protein + half DNA

nucleosome

DNA wound tightly around proteins called histones

histones

proteins

breakdown/steps of condensed DNA

1. DNA wounds around proteins called histones become a nucleosome (beads on a string)

2. then, other proteins are added to each wound that allow for nucleosomes to be tightly packed

3. then, packed even more tightly into a fiber called chromatin (30-nm fiber)

4. lastly there is chromatin folding with loops of 30-nm fibers

chromatin can exist in two structural/function states

heterochromatin and euchromatin

heterochromatin

highly condensed, gene-poor, and transcriptionally silent (genes not accessible)

euchromatin

less condensed, gene-rich, and more easily transcribed (the genes are accessible)

how does the structural state of chromatin impact DNA’s function?

• decondensed: for access

• condensed: for storage

• not just for storage: the cell decides which genes need to be accessed to do the specific work at a particular time

• controls access to regions of DNA for: replication, expression, and repair

structure of nucleosomes

• DNA: about 147 base pairs are wrapped around around the protein core (1 and 2/3rds of the way); negative charge

• protein core: octomer of histone proteins; positive charge

• histones play an important role in condensing chromatin

• opposites attract —> tight interaction

chromatin remodeling enzymes

modify histone tails to impact chromatin packing

histone H3

1. methyltransferase modifies histone tails by transferring methyl groups onto histone tails (histone H3 for this example). this makes the tails for accessible since they become popped up.

2. the methylated histone tails bind to protein HP1. The tails are now available for something to attach to them after being methylated

3. HP1 oligomerization (many units come together) brings nucleosomes together. HP1 binds to itself which pulls all nucleosomes to on another and condenses

methyltransferase

transfers enzyme methyl groups

does methylation of histone tails increase or decrease gene expression

increase in condensation —> decrease in gene expression (less accessible)

You are studying HP1, a protein involved in condensing chromatin, by making mutations in HP1 and studying their effect on the formation of heterochromatin in a eukaryotic cell. Predict what effect each mutation would have on the condensing of chromatin.

Compared to normal (“wildtype”) HP1, a mutant version of HP1 that oligomerizes more strongly would":

A. enhance heterochromatin formation

B. inhibit heterochromatin formation

C. have no effect on heterochromatin format

A

You are studying HP1, a protein involved in condensing chromatin, by making mutations in HP1 and studying their effect on the formation of heterochromatin in a eukaryotic cell. Predict what effect each mutation would have on the condensing of chromatin.

Compared to normal (“wildtype”) HP1, mutant version of HP1 that cannot bind H3L9Me3 would:

A. enhance heterochromatin formation

B. inhibit heterochromatin formation

C. have no effect on heterochromatin format

B

Modifications

• there are three types of modifications cells use on their histone tails to regulate chromatin packing: methylation, phosphorylation, acetylation

• modifications are site-specific, not random

phosphorylation

addition of phosphate group, -PO4

acetylation

addition of acetyl group, -COCH3, catalyzed by histone acetyltransferases. “opens” the chromatin and increases gene expression (DNA is more accessible)

methylation

addition of methyl group, -CH3

deacetylation

removal of acetyl group, catalyzed by histone deacetylases

Based on the chemistry of the acetylation of lysine, would you expect this histone tail acetylation to tighten or loosen the interaction between the histone and the DNA wrapped around it?

it would loosen the interaction between the histone and the DNA wrapped around it. lysine is positive, so with the addition of the acetyl group, the DNA (which is negative) and the lysine (the histone) will lose attraction between each other due to a lack of opposite attraction

cytosine methylation (CpG methylation)

• modifications can also occur on the DNA molecule itself.

• methylation on cytosines that are followed in the same DNA strand by a guanine in the 5’ to 3’ direction. (not to be confused with C=G base pairs across the two DNA strands.)

• This can enhance chromatin condensation. methylated DNA can serve as a binding site for other proteins, including deacylation and other chromatin remodeling proteins that condense chromatin.

is cytosine methylation heritable? (think about semi-conservative replication of DNA)

• since DNA is mirrored across both strands, it will be the correct bases when copied, allowing it to be methylated.

• half is new and half is old, if the methyl group is on the old strand, it will be passed down.

DNA accessibility in cytosine methylation

• cytosine methylation can make DNA less accessible.

• methylated DNA can serve as a binding site for other proteins that condense chromatin

epigenetics

the study of heritable changes in gene expression that do not involve changes to the underlying DNA sequence (a change in phenotype without a change in genotype)

active (open) chromatin

• unmethylated cytosines (white circles)

• acetylated histones (Ac)

• unmethylated histone tails

silent (condensed) chromatin

• methylated cytosines (red circles)

• decetylated histones

• methylated histone tails

DNA structure

the structure of DNA provides a mechanism for its function as the molecule that encodes hereditary information

inheritance of traits

DNA is easily reproducible and stable

evolution of traits

DNA sequence is easily changeable and modular

topoisomerase

unwinds the double helix of DNA

helicase

separates double-stranded DNA into single-stranded DNA (unzips)

single stranded binding proteins

prevent complementary base-pairing at fork, keep DNA from reconnecting (keeping them separate)

primase

starts new strand by synthesizing short RNA sequence called a “primer”

DNA polymerase

• starting at primer, elongates DNA polymer, catalyzing covalent bonds of sugar-phosphate backbone

• adds complementary nucleotides to the free 3’ hydroxyl group of a growing DNA strand to the incoming 5’ phosphate group

• uses deoxyribonucleotides to fill in the gaps left by removal of RNA primers

leading strand

continually synthesized toward replication fork

lagging strand

synthesized in sections

the herpes drug acyclovir works by inhibiting the synthesis of new viral DNA. acyclovir resembles the base guanine but does not contain the full 5-carbon ring of the sugar backbone.

predict how acyclovir’s structure blocks the synthesis of new viral DNA.

because DNA polymerase needs the 5-carbon sugar and phosphate group to synthesize DNA, it cannot do that because there no 5-carbon group/3’hydroxyl group, so another base cannot be added after guanine

synthesis of DNA from lagging strand

1. primase adds RNA primers

2. DNA polymerase starts at the primer and elongates DNA polymer, catalyzing covalent bonds of a sugar-phosphate backbone (sections are called Okazaki fragments)

3. exonuclease removes RNA primer

4. DNA polymerase fills in the gaps

5. DNA ligase catalyzes the covalent bonds of the sugar-phosphate backbone, sews the two fragments together

exonuclease

removes RNA primer

DNA ligase

catalyzes covalent bonds of sugar-phosphate backbone of adjacent pieces of DNA (sews together)

where does DNA replication begin and end?

at the replication origin, end when it eventually meets another bubble or when it reaches the end of the molcule

replication forks

bubbles on either side of the origin. template DNA on the outside and newly-synthesized DNA on the inside

is there only one replication fork?

there can be multiple replication forks along the molecule of DNA

leading and lagging strands in the replication fork

• the leading and lagging strands are relative to the replication fork

• one strand can be both leading & lagging, depending on what side of the strand the fork its

• because there can be multiple replication forks along on DNA molecule, the leading and lagging strands will eventually meet and merge (replication ends here or when replication meets the end of the DNA)

PCR

• polymerase chain reaction

• DNA replication happening in a test tube

• making copies/amplifying genes of interest

• primers: uses DNA primers instead of RNA primers

• Taq polymerase: elongates new DNA

• no replication fork

• no lagging strand

denaturing

PCR uses heat to make DNA copies. enough heat to break apart the strands but not enough to break the backbone (unzipping w/heat)

which components of cellular replication machinery are no longer necessary in PCR because heat is used to separate the DNA strands?

helicase

annealing

temperature is lowered and DNA primers attach to DNA template strand

which components of the cellular replication machinery are no longer necessary in PCR because scientists design DNA primers to initiate replication?

• primase: PCR uses DNA primers instead of RNA primers

• exonuclease: gets rid of RNA primers, but we’re using DNA primers which gets incorporated with the strand since it’s already DNA

• ligase: don’t need to be sewn up because DNA primers are staying

extension

synthesize new strand, Taq polymerase adds nucleotides to the annealed primer, extension/elongation of strand

PCR cycling

many cycles = even more copies

two methods of detection of PCR products (the DNA sueqences of interest)

by fluorescence and by gel electrophoresis

fluorescence

• use primers specific to parts of genome of interest

• use fluorescence to measure how many copies of the genomes of interest are present

• sensitive enough to detect only 1 virus (in the example of detection of SARS-CoV-2 from nasal swabs)

gel electrophoresis

• use primers specific to gene of interest

• visualize PCR products as a band in a gel