BIOL 201 Chapter Notes - Chapter 1,4,10-15: Dna Polymerase Iii Holoenzyme, Dna Clamp, Okazaki Fragments
25 May 2018
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DNA REPLICATION PROCESS
A helicase hydrolyzes ATP to start unwinding at the replication fork. A single stranded binding
protein binds to single stranded DNA to prevent reannealing. DNA gyrase, untwists the DNA in
front of the replication fork. Topoisomerases are enzymes that participate in the over winding or
underwinding of DNA. DNA polymerase is an important enzyme that carries individual
nucleotides to the site of replication. DNA polymerase builds the daughter strand by matching
nucleotides to their complementary bases on the parent strand. But the action of DNA
polymerase is not always continuous because the DNA backbone runs in opposite directions. The
DNA strands running in opposite directions were named according to the carbons in the sugar
ring. One end is called the 3’ end and the other is called 5’ end. So, on any complete molecule of
DNA, one stand will run from 3’ to 5’ and the other will run from 5’ to 3’. But, DNA polymerase
only works in the 3’ to 5’ direction. For one of the parental strands of DNA, replication occurs
normally with DNA polymerase working continuously to add on the nucleotides. But for the
other strand, which runs in the 5’ to 3’ direction, DNA polymerase cannot replicate that way.
The how is the other parent strand replicated?
The first strand is called the leading strand which runs in the 3’ to 5’ direction towards the fork
and it’s able to be replicated continuously by DNA polymerase. Polymerase III is responsible for
the synthesis of leading strand. The other strand, which runs in the 5’ to 3’ direction is called the
lagging strand and this one is replicated discontinuously. What does discontinuous and
continuous mean? The leading strand’s free end is a 3’ end and the end that is nearest to the
replication fork is the 5’ end, so DNA polymerase can simply start at the free end, working in the
3’ to 5’ direction and run continuously towards the replication fork. The lagging strand synthesis,
however; produces short Okazaki fragments. The steps in lagging strand synthesis include: the
generation of an RNA primer by primase. Polymerase III directs template DNA synthesis, after
which the Polymerase III is released from the lagging stranded due to the presence of double
stranded DNA. since Pol III has no 5'-3' exonuclease activity, it halts when it meets the RNA
primer from the previous priming event. A DNA clamp, also known as a sliding clamp, is
a protein fold that serves as a process-promoting factor in DNA replication. As a critical
component of the DNA polymerase III holoenzyme, the clamp protein binds DNA
polymerase and prevents this enzyme from dissociating from the template DNA strand. The beta
clamp is a specific DNA clamp and a subunit of the DNA polymerase III holoenzyme found in
bacteria. Two beta subunits are assembled around the DNA by the gamma subunit and ATP
hydrolysis; this assembly is called the pre-initiation complex. After assembly around the DNA,
the beta subunits' affinity for the gamma subunit is replaced by an affinity for the alpha and
epsilon subunits, which together create the complete holoenzyme. DNA polymerase III is the
primary enzyme complex involved in prokaryotic DNA replication. RNase H removes most of
the RNA primer, Pol I bind to the small gap. Uses 5'->3' exonuclease and 5'->3' polymerase to
remove the remaining RNA primer. DNA ligase then seals the break in the phosphodiester bond,
joining the newly synthesized Okazaki fragment, with its RNA replaced by DNA, to the
previously replicated DNA.
Chapter 1
Seven characteristics that define something as being “alive”
Cellular organization: each living thing is composed of cells which carry out the basic activities of life.
Cells are enclosed in a membrane that separates it from its environment.
Ordered complexity: all living things are composed of complex structures that are highly ordered. Many
non-living things are also complex but their level of complexity is not as high as those of living things.
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Sensitivity: all organisms respond to stimuli, e.g. plants grow in the direction of sunlight or you move
your hand away from a lighted candle when you feel the burn.
Growth, development, and reproduction: all organisms grow and reproduce. The parents have heredity
molecules that are passed down from one generation to another.
Energy utilization: all organisms require energy to carry out daily activities. The energy we get from our
diet is used by us to carry out daily activities.
Homeostasis: all organisms maintain constant internal conditions. For example, the internal temperature
of the body would remain the same regardless of the outside temperature.
Evolutionary adaptation: all organisms interact with living organisms and their non-living environment
in ways that influences their survival and they adapt to their evolving environment.
Hierarchical level of living organisms:
Cellular level:
Atoms → molecules → cells → organelles → membrane bounded cells
Organismal level:
Similar cells → tissues → organs (brain) → organ systems (nervous system)
Populational level:
different organ systems → organism → population (a group of organisms of the same species living in the
same place) → species (same population)
ecosystem level:
populations + interaction with their environment = ecosystem
biosphere: the entire planet can be thought of as an ecosystem that we call the biosphere.
Emergent properties: emergent properties are those properties that arise when small structures combine
to function together as one system. For example, cells make tissues, tissues make organs, organs make
organ systems like nervous system, digestive system, respiratory system etc. one cannot determine the
function of a nervous system just by looking at one cell because that one cell doesn’t exhibit the
properties of the nervous system.
Novel properties that arise from the way in which components react. Emergent properties often cannot be
solely deduced from the knowledge of the individual component.
Inductive vs deductive reasoning
Deductive reasoning: One applies general principals to predict specific results. For example, all
mammals have hair, but if scientists come across an animal that does not have hair then they conclude
that, that animal is not a mammal.
GENERAL STATEMENTS → SPECIFIC CASES
Inductive reasoning: in inductive reasoning, the logic flows backwards. From specific to general.
Inductive reasoning uses specific results from observations and construct general scientific principles. For
example, if poodles have hair and terriers have hair, and every dog that that you observe has hair, then
you can conclude that all dogs have hair.
SPECIFIC CASES → GENERAL STATEMENTS
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How scientific investigations proceed:
• Observations are made about subjects that raise questions. These questions are then answered in
terms known as hypothesis.
• All the hypothesis are then tested through experiments which enables us to further reject a few of
them.
• Predictions are made based on these hypotheses which are further tested through experiments.
• If the experiments support the hypothesis then the predictions support the hypothesis as well. If
the predictions do not support the hypothesis, then it needs to be either rejected or modified
accordingly.
Example: when nutrient broth was left untouched in open air, it became contaminated. The two
hypothesis that supported this contamination were spontaneous generation and germ hypothesis.
Spontaneous generation meant that there were inherent properties in organic molecules that led
the spontaneous generation of life. The germ hypothesis suggested that there were preexisting
microorganisms present in the air that contaminated the air.
The definite experiment was conducted by louis Pasteur who created a flask with a curved neck.
The curved neck could be exposed to air but all the germs settled down in the neck or curve of the
flask whereas leaving the broth non-contaminated. But as soon as the curved neck was broken
off, the broth inside the flask got contaminated as well thus supporting the germ hypothesis.
*A hypothesis can be disproved but it cannot be proved correct.
Observations → hypothesis → experiments → rejected hypothesis → predictions based on
hypothesis → experiments on predictions → hypothesis rejected or accepted.
Reductionism:
Reduction is the breakdown of complex systems into smaller parts to understand them better. Even
though reductionism has benefited scientists greatly in understanding cellular metabolism, it has its
limitations as well. Two of these limitations are:
• Enzymes do not work the same in isolated environments as they do in their normal cellular
context.
• The complex interworking of interconnected functions lead to emergent properties that cannot be
noticed while studying parts of a complex system. For example, the function of a ribosome is to
synthesize proteins. But this function cannot be predicted if we just look at RNA and protein
alone which make up the ribosome.
What is scientific theory?
Scientific theory is a theory that is supported by vigorous experiments and results. For example, the
quantum theory of physics, the theory of gravity etc. In the field of science, theory will always be
referenced as an accepted general principle or a body of knowledge. As compared to the definition of
theory which the public usually refers to is “a guess or a lack of knowledge.”
Basic vs. applied research: In basic research, scientists extend the boundaries of what they know. These
scientists usually work at universities and their research is funded through grants. The information
generated through basic research is then used as a foundation to applied research. The scientists
conducting applied research are usually employed in industry where they test food additives, create new
drugs or test environmental quality.
What is descriptive biology?
Descriptive science does not involve production hypothesis and testing it. Scientists are just trying to
explain something as completely as they can.
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Document Summary
A helicase hydrolyzes atp to start unwinding at the replication fork. A single stranded binding protein binds to single stranded dna to prevent reannealing. Dna gyrase, untwists the dna in front of the replication fork. Topoisomerases are enzymes that participate in the over winding or underwinding of dna. Dna polymerase is an important enzyme that carries individual nucleotides to the site of replication. Dna polymerase builds the daughter strand by matching nucleotides to their complementary bases on the parent strand. But the action of dna polymerase is not always continuous because the dna backbone runs in opposite directions. Dna strands running in opposite directions were named according to the carbons in the sugar ring. One end is called the 3" end and the other is called 5" end. Dna, one stand will run from 3" to 5" and the other will run from 5" to 3". But, dna polymerase only works in the 3" to 5" direction.
