TA: Alberto Lopez
FALL 2015
Discussion D4
Lectures 19-20 Study Guide
I have organized some terms and topics that I think are important. This does not mean that other topics
mentioned during lecture or in the book will not be tested. This guide is meant to clarify and emphasize certain
points, NOT to list everything you need to know. I will focus on tying things together across lectures, and giving
real-life examples of the biological principles that we are learning. Details that I include that I think will be
helpful, but that you don’t need to know, I will write in green. Questions to think about I will write in blue.
Lecture 19: DNA- The Molecular Basis of Inheritance
Terminology
1. Transformation: a cell incorporates DNA from another source, leading to a change in its genotype and
phenotype
2. Bacteriophages / phages: viruses, which are basically just DNA / RNA surrounded by a few proteins
3. Double helix: basic structure of DNA. Two polynucleotide strands coiled into a helix.
In a DNA double helix, where are the covalent bonds? Hydrogen bonds?
4. Anti-parallel strands: the two DNA strands run in opposite directions, and are complementary to one
another
How can you tell which end is the 5’ end and which is the 3’ end? What specific functional groups can
you look for?
5. Base pairing: purines bind to pyrimidines; A binds to T, C binds to G
6. Origin of replication: point at which the two DNA strands separate to form the replication bubble
7. Template strand: during DNA replication, each parent strand is used as a guide to build a new daughter
strand on
Key concepts
DNA is the genetic material
1. Frederick Griffith; Experiment with bacteria:
a. 2 strains of bacteria, one lethal (“S”) and one not (“R”)
b. If you mix dead S cells with live R cells, the R cells become lethal
c. IMPORTANTLY, the next generation of R cells are now lethal, too, indicating that lethality was a
heritable change
d. In subsequent experiments, researchers took either DNA, RNA or protein from the dead S cells,
and found that only the DNA was able to transform the R cells to become lethal
2. Hershey and Chase; Experiment with virus:
a. Phage = virus that infects bacteria = DNA + protein
b. Label either proteins (radioactive sulfur) or DNA (radioactive phosphorus)
c. Measure radioactivity in infected cells: do the cells contain radioactive sulfur or phosphorus?
d. The cells contained radioactive phosphorus, indicating that the phages were infecting bacteria
by injecting DNA
e. Hershey and Chase concluded that DNA, not protein, functions as the genetic material of phage
T2. Convinced scientists that DNA was the heredity material
3. The genetic information encoded in DNA is stored as sequences of nucleotide bases, of which there are
four varieties: adenine, guanine, cytosine and thymine
Base pairing occurs between a purine and a pyrimidine
1. Purines = A and G, pyrimidines = C and T
2. Purines always bind to pyrimidines
Modified from materials prepared by Carley Karsten, 2013
1
TA: Alberto Lopez
FALL 2015
Discussion D4
3. A always binds to T, and C always binds to G
 Thus, the amount of A in an organism is always equal to the amount of T, and the amount of C
equals the amount of G
4. A=T pairing uses two hydrogen bonds, while C≡G pairing uses three hydrogen bonds
What is special about regions of DNA that are rich in As and Ts?
DNA replication is semi-conservative
1. The two strands of DNA are complementary, meaning that if you know one you can figure out the
other. This is because of base pairing. So if you know that one strand looks like this: 5’ –
CGAATAGCTGA – 3’, you can easily figure out that the other strand looks like this: 3’ –
GCTTATCGACT – 5’
2. During replication, the parent strands separate and a new daughter strand is added to each one,
resulting in two complete double helices
3. Each daughter molecule is made up of one parental strand and one new strand
This is really awesome when you think about it. Remember that normal cells divide dozens of times
throughout their lives. This means that they have to replicate their DNA a lot. By building new strands
on top of old strands, it makes things faster and more accurate. Compare tracing a picture to drawing
one freehand. WAY easier to trace it.
4. Important experiment that showed us this:
a. Grow bacteria in heavy (15N) and then light (14N) isotopes of nitrogen
b. Allow bacteria to replicate twice in the light isotope, and measure the DNA density of the two
daughter generations
c. Looking at the second daughter generation, there are three possible outcomes (draw this out or
look at the picture on the slide if you’re confused):
i. Parental strands will come back together after replication, so you’ll have a heavy band
(parent DNA, all 15N) and a light band (daughter DNA, all 14N)
ii. Parental strands persist as part of the daughter cell DNA, so you’ll have an intermediate
band (DNA is half-parent and half-new, so a mix of 14N and 15N) and a light band (all 14N)
iii. Every daughter cell has an equal mixture of parental and new DNA (all bands would be
the same density, because they’re all made of equal parts 14N and 15N)
d. Results: one light band and one intermediate band! This showed scientists that replication was
semi-conservative.
Lecture 20: DNA Replication and Repair
Terminology
1. Helicase: separates the two parent strands (“unzips the genes”)
2. Topoisomerase: unwinds DNA ahead of the replication fork. If it didn’t do this, the DNA would get really
coiled up as helicase pushes the replication fork forward, and the DNA would break.
3. Single strand binding protein: keeps separated strands from coming back together. Since the strands are
complementary, they really like being together. Without SSBPs, they would stick together all the time
and primase / polymerase would never be able to get in!
4. RNA primase: adds RNA primer to single-stranded DNA
Why do we *need* a primer?? (Hint: why does replication only occur 5’ to 3’?)
5. Nucleoside triphosphate: floating free in the nucleus, these are made of nucleotides with two extra
phosphate groups
6. DNA polymerase: adds new nucleotides to single-stranded DNA, removes RNA primers, and fixes basepairing mistakes
Modified from materials prepared by Carley Karsten, 2013
2
TA: Alberto Lopez
FALL 2015
Discussion D4
7. Telomeres: long strings of nonsense DNA at the ends of chromosomes
Key Concepts
DNA replication occurs in the 5’ to 3’ direction
1. This means that the new DNA strand is created in the 5’ to 3’ direction (this can get confusing, since it is
being added to the parent strand in the 3’ to 5’ direction… be sure you understand this distinction)
2. Why?  Because new nucleotides can only be added to the 3’ (-OH) end of the sugar-phosphate
backbone. If DNA polymerase doesn’t see an –OH, it won’t work.
Many enzymes involved in replication: described here in the order they appear
1. Helicase inserts itself into the replication bubble and pushes it forward, separating the two DNA strands
from one another
2. Topoisomerase prevents the DNA ahead of the replication fork from winding too tightly. It does this by
cutting out chunks of DNA, untwisting them, and then sticking them back where they belong.
3. RNA primase adds a few (5-10) RNA nucleotides to the parental DNA strand. The 3’ end of these
nucleotides is what attracts DNA polymerase.
Why RNA and not DNA? As we will see next week, transcription (the process of converting DNA to RNA)
does not require primers to begin. It is easier to add RNA from scratch than to add DNA.
4. DNA polymerase picks up free nucleoside triphosphates and adds them to the 3’ ends of existing
nucleotides. In the process, the two extra phosphate groups are lost and a phosphodiester bond is
formed. Remember: phosphodiester bond = sugar – phosphate group – sugar.
Leading vs. lagging strands
1. Leading strand = easy. Starting at the origin of replication, DNA polymerase just keeps adding
nucleotides as far as it can (5’ to 3’). Moves toward the replication fork.
2. Lagging strand = complicated. Starting a little bit after the origin of replication, DNA polymerase adds
nucleotides backwards, away from the replication fork.
a. IMPORTANT: “backwards” only means away from the replication fork. Nucleotides are still being
added 5’ to 3’. They always are.
b. Since we’re moving away from the replication fork, replication has to start over every time the
fork moves farther away. This results in little chunks of DNA called Okazaki fragments. These
fragments are later glued together by the enzyme DNA ligase.
If you can’t remember that ligase is the enzyme that connects DNA fragments together, it might
help to think of ligaments, which are the connective tissues in our bodies.
3. Keep in mind that replication begins from a replication bubble, and results in TWO replication forks
moving in opposite directions.
You’re used to seeing images like this:
And it’s easy to forget that it’s actually happening like this:
Modified from materials prepared by Carley Karsten, 2013
3
TA: Alberto Lopez
FALL 2015
Discussion D4
So, for every origin of replication, you have two leading strands and two lagging strands. As each
Okazaki fragment catches up with the last one, DNA ligase seals them together. Likewise, when the last
Okazaki fragment catches up with the end of the other leading strand, DNA ligase seals them together
too.
Based on the diagram above, where are the 5’ and 3’ ends of the DNA strands?
Replicated DNA is proofread and repaired
1. DNA polymerase “proofreads” new DNA to be sure everything matches up correctly (i.e. As are only
bound to Ts, etc.)
2. Mismatch repair: when bases are paired wrong, they are replaced by the correct bases. Only needs to
cut out the base, backbone is unaffected.
3. Nucleotide excision repair: when DNA is damaged, nucleotides are cut out and replaced by new ones.
Involves cutting through the backbone and replacing everything.
Telomeres are sequences of nonsense DNA at the end of chromosomes
1. Lagging strands can’t replicate the very ends of DNA strands. As a result, you lose a few base pairs every
time you replicate your DNA!! Thankfully, there’s a solution: telomeres. These are extra base pairs at
the ends that don’t code for anything at all. So when they’re lost, it doesn’t matter.
2. However, even with the telomeres, with enough cell divisions they will disappear and you’ll start losing
important DNA. Telomerase is an enzyme that prevents telomere shortening in germ cells only, to
ensure that you won’t pass on already-shortened chromosomes to your offspring.
Modified from materials prepared by Carley Karsten, 2013
4