CS&B - Biochemistry - DNA Structure, Replication, Repair Flashcards
Euchromatin is known as ‘beads on a string’ due to its arrangement as nucleosomes connected by linker DNA. These fibers are commonly referred to by their width.
What width does euchromatin have?
11 nanometers
Approximately, how many basepairs (bps) are found twice-wound around the histone octamer?
And how many are found in the linker (spacer) DNA between nucleosomes?
146 bps;
90 bps
The weak forces connecting DNA to histones are primarily produced by electrical interactions between what structures?
DNA phosphate groups (-) and arginine and lysine side chains (+)
What are the proteins making up the histone octamer?
H2a (x2)
H2b (x2)
H3 (x2)
H4 (x2)
What histone protein is not part of the histone octamer?
H1
One fiber of euchromatin is ___ nm in width.
One fiber of heterochromatin is ___ nm in width.
11;
30
What portion of the nucleosome is the site of most modifications that allow for gene activation and silencing?
The histone tails
What major nucleosome modifier is responsible for decondensation (unwinding) of chromatin?
What effect will this have on the genes involved?
Acetylation;
increased gene expression
How does acetylation of the histone tails (specifically lysine residues) cause decondensation of the chromatin?
It neutralizes some histone positive charge
What enzyme is responsible for histone acetylation?
What enzyme is responsible for histone deacetylation?
Histone acetyltransferases (HATs);
histone deacetylases (HDACs)
Histone acetyltransferases (HATs) have what effect on chromatin?
Histone deacetylases (HDACs) have what effect on chromatin?
Decondensation (11 nm fibers);
condensation (30 nm fibers)
Histone acetyltransferases (HATs) have what effect on gene expression?
Histone deacetylases (HDACs) have what effect on gene expression?
Increased gene expression;
gene silencing
What effect does methylation typically have on chromatin?
Gene silencing
How does histone methylation cause gene silencing?
By preventing acetylation
What is the typical effect of histone phosphorylation?
To promote chromatin decondensation (unwinding)
Which histone modifications will typically result in chromatin decondensation (unwinding) and increased gene expression?
Acetylation (by HATs);
phosphorylation
Which histone modifications will typically result in chromatin condensation (coiling) and gene silencing?
Deactylation (by HDACs);
methylation
For what are SWI/SNF responsible in gene expression?
How?
Regulation of chromatin decondensation;
through HAT function
For what is H1 responsible in gene expression?
How?
Facilitating DNA condensation;
through HDACs
Which activators regulate HAT function and chromatin decondensation?
Which activators regulate HDAC function and chromatin condensation?
SWI/SNF;
H1
What is the function of the MECP2 protein?
To bind methylated DNA (not methylated histones)
and recruit HDACs
DNA methylation (as opposed to histone methylation) will result in activation of what protein and what effect?
The MECP2 protein;
HDAC recruitment;
chromatin condensation
What is the shortest phase of the cell cycle?
What phase is significantly shortened in rapidly dividing (e.g. tumor) cells?
M;
G1
How many chromosomal autosome pairs are there?
How many chromosomal sex pairs are there?
22;
1
What stain is often used in karyotyping so different chromosomes can be distinguished from one another?
What segments are marked using this staining process?
G-banding;
A-T rich sequences
What are some notable defects that can potentially be observed on karyotyping?
Chromosomal breaks, deletions, duplications, translocations, aneuploidy, and more
Define aneusomy.
What are the three types?
Aberrant number of chromosomes;
nullisomy, monosomy, trisomy
Define euploidy.
Correct number of chromosome pairs
What is the term if an embryo is missing both chromosomes of one chromosomal pair?
What is the term if an embryo is missing one chromosome of a chromosomal pair?
What is the term if an embryo has both chromosomes of one chromosomal pair?
What is the term if an embryo has an extra chromosome in addition to one chromosomal pair?
Nullisomy (-2);
monosomy (-1);
euploidy (0; just right);
trisomy (+1);
Describe chromosomal structure using the following terms:
chromatid
coil
11 nm fiber
loop
rosette
30 nm fiber
What enzyme cuts specific palindromic DNA sequences?
Restriction endonucleases
DNA of interest can be recombined into a plasmid after cutting palindromic sequences on both using what type of enzyme?
What enzyme then seals their exposed palindromic sequences?
What then happens in the bacteria containing this recombined DNA?
An endonuclease;
DNA ligase;
replication (of the cell and DNA)
How can a radio-labeled probe be used to identify a single gene of interest in a genome?
Heat the cell (denaturing);
add a radio-labeled probe;
reduce the heat (the probe anneals to the gene of interest)
What in particular can in-situ hybridization be used to analyze?
Gene expression (through antibodies that show mRNA and/or protein expression)
What specialized base pairs are useful for Sanger DNA sequencing? What is important about the base pair insertions?
Dideoxynucleotides (ddATP, ddGTP, ddCTP, ddTTP);
one must attach after each base pair of the target sequence, creating sequences of all lengths from the full sequence to only one base pair
What is PCR good for?
Amplifying DNA sequences using a system of primers, heating, and Taq polymerases
How do we know that genetic polymorphisms are not simply new mutations?
They are present in ≥1% of the population
Per every 1 billion base pairs copied, how many mutations occur?
~1
What general term can be used to describe DNA replication in regards to the way older DNA strands are distributed amongst newer strands as replication occurs?
It is semi-conservative
True/False.
DNA is both antiparallel and complementary.
True.
What does it mean that DNA replication is semi-conservative?
Each new DNA duplex model is composed of one parent strand and one daughter strand
DNA origins of replication are characterized by a richness of what type of base pairing?
A-T pairings
Where on a replication bubble would the origin of replication be found?
Where would the replication forks be found?
In the middle of the bubble;
at the ends
All DNA replication occurs in what direction?
5’ to 3’
Which strand of DNA replication is the leading strand?
Which strand of DNA replication is the lagging strand?
The strand in which replication is continuous;
The strand in which replication is discontinous
What structure is used to provide a 3’-hydroxyl group so that DNA replication can initiate after the strands are denatured from one another?
An RNA primer
What enzyme ‘unwinds’ DNA helices from one another?
What enzyme releases the tension this creates by snipping and rejoining the strands?
What proteins prevent reannealing of the strands?
Helicase;
topoisomerase;
single-stranded binding proteins
In-vitro DNA replication mutations are 1 out of every 10,000 base pairs. In-vivo human replication mutations are 1 out of every 1,000,000,000 base pairs.
What explains this discrepancy?
Extensive DNA repair systems
What bacterial DNA polymerase is the main one synthesizing new strands?
DNA polymerase III (bacterial)
What bacterial DNA polymerase replaces RNA primers with DNA?
DNA polymerase I (bacterial)
What bacterial DNA polymerases are most involved in DNA repair?
DNA polymerase I and II (bacterial)
How does DNA polymerase I (bacterial) remove RNA primers to be replaced with DNA?
3’ to 5’ exonuclease activity
What enzyme connects Okazaki fragments?
DNA ligase
How many replication origin points do eukaryotes have in each contiguous piece of DNA?
(1 or many?)
Many
Where is the hydroxyl group that the triphosphate bond attacks in DNA polymerization?
(I.e. hydroxyl groups of what prime are attacked by phosphates of what prime?)
3’-hydroxyl
5’-phosphate
For what is eukaryotic DNA polymerase Alpha useful?
Synthesizing short stretches of DNA, works with RNA primase to make RNA primers
For what is eukaryotic DNA polymerase Delta useful?
Takes over after RNA primers; lagging strand synthesis (but also can be leading)
For what is eukaryotic DNA polymerase Epsilon useful?
Leading strand synthesis (but also can be lagging)
For what is eukaryotic DNA polymerase Beta useful?
DNA repair
For what is eukaryotic DNA polymerase Gamma useful?
Mitochondrial DNA replication
Which eukaryotic DNA polymerase is responsible for aiding in the synthesis of RNA primers and short stretches of DNA that follow?
DNA polymerase alpha
Which eukaryotic DNA polymerases are processive?
Delta, epsilon
Which eukaryotic DNA polymerases are involved in nuclear DNA replication?
Alpha, delta, epsilon
Which eukaryotic DNA polymerase is involved in DNA repair?
Beta
Which eukaryotic DNA polymerase is involved in mitochondrial DNA replication?
Gamma
Which eukaryotic DNA polymerase is mainly involved in nuclear DNA replication of the leading strand but can also contribute to the lagging?
Epsilon
Which eukaryotic DNA polymerase is mainly involved in nuclear DNA replication of the lagging strand but can also contribute to the leading?
Delta
What enzyme is able to ‘read’ RNA in order to synthesize new DNA?
What types of infectious agents are likely to have this enzyme?
Reverse transcriptase;
retroviruses (e.g. HIV)
What infectious agent uses the HSV DNA polymerase?
Herpes simplex virus
DNA synthesis occurs in what direction?
5’ to 3’
What bacterial enzyme removes and replaces the RNA primers of DNA replication?
How?
DNA polymerase I;
5’ to 3’ exonuclease activity
What eukaryotic enzymes remove and replace the RNA primers of DNA replication?
How?
RNAseH and Fen1;
5’ to 3’ exonuclease activity
DNA ligase forms what type of bond?
3’ to 5’ phosphodiester bonds
Which bacterial enzyme is able to perform both 5’ to 3’ DNA synthesis and 3’ to 5’ exonuclease activity?
DNA polymerase I
DNA proofreading by bacterial DNA polymerase I occurs in what direction?
3’ to 5’
How is bacterial DNA polymerase I able to perform both 5’ to 3’ synthesis and 3’ to 5’ exonuclease activity?
There are two separate active sites on the enzyme
What enzyme fills in the space left behind by RNA primers at the end of genetic sequences?
Why is this important?
Telomerase (TTAGGG);
to protect against chromosomal shortening
Why is telomerase needed in DNA replication?
The final Okazaki fragment of the lagging strand has a gap at the end where no RNA primer 3’-hydroxyl is be placed as the space is shortened
Which enzyme(s) is(are) responsible for processive DNA synthesis in bacteria?
Which enzyme(s) is(are) responsible for processive DNA synthesis in eukaryotes?
DNA polymerase III;
DNA polymerase delta, DNA polymerase epsilon
DNA is synthesized in what direction?
The template used for synthesis is read in what direction?
5’ to 3’;
3’ to 5’
What are a few exogenous sources of DNA damage?
Ionizing radiation;
UV rays;
environmental chemicals
What are a few endogenous sources of DNA damage?
Reactive oxygen species (ROS);
uncorrected errors of replication
Chemical and ROS damage to DNA often results in what sort of structural changes?
UV damage to DNA often results in what sort of structural changes?
Depurination, deamination;
thymidine dimers
Deamination of cytosine leads to:
Uracil
What are three forms of DNA repair?
(the three ‘excision repairs’)
Base excision repair
Nucleotide excision repair
Mismatch excision repair
Deamination of 5-methylcytosine leads to:
Thymine
How are basepairing errors and other minor DNA errors corrected?
What is a common example of a new basepairing error?
Base excision repair;
5-methylcytosine is deaminated to thymine
(C to T)
How are thymidine dimers and other large DNA lesions corrected?
Nucleotide excision repair
What is the most common form of basepair error?
What type of DNA repair mechanism would address this?
Cytosine to thymine mutations
(and subsequent T-G base pairs);
base excision repair
How much of the DNA strand is removed in base excision repair?
Just the errant base
How much of the DNA strand is removed in nucleotide excision repair?
Often several nucleotides in sequence
How much of the DNA strand is removed in mismatch excision repair?
Often several nucleotides in sequence
What two enzymes of nucleotide excision repair will detect large bulges and defects in the DNA?
XP-C or Rad23B
(both part of a genomic scanning complex)
After XP-C and Rad23B detect a large lesion, what are the next steps of nucleotide excision repair?
Helicase unwinds the strand;
endonucleases remove the offending portion
(DNA polymerase and ligase then refill the portion)
What are the two endonucleases of nucleotide excision repair?
XP-F and XP-G
What is the rate-limiting enzyme of nucleotide excision repair?
What gene codes for it?
XP-F;
ERCC1
Cisplatin is used to damage DNA and induce apoptosis.
What defense mechanism is fixing this damage?
Cells overexpressing what gene will be resistant to cisplatin?
Nucleotide excision repair;
ERCC1 (XP-F gene)
What type of DNA repair mechanism catches uncorrected errors of replication?
Mismatch excision repair
What is the difference between nucleotide excision repair (NER) and mismatch excision repair (MER)?
NER corrects large defects (e.g. thymidine dimers);
MER corrects errors of replication (e.g. C-A pairing)
Which of the three forms of DNA excision repair (base, nucleotide, mismatch) involve(s) helicase, endonucleases, polymerase, and ligase in removing sequences of DNA (i.e. more than one nucleotide) and refilling them?
Nucleotide, mismatch
(base only removes a single, solitary base)
What are the two types of gene (based on whether the gene is virtually always active and needed or not)?
Constitutive;
regulated
What is a constitutive gene?
(as opposed to regulated)
A gene that is always active in virtually all cells
(needed for basic cell survival)
What is a regulated gene?
(as opposed to constitutive)
A gene that is only needed sometimes;
they are turned on and off according to cellular needs/signals
Constitutively expressed genes are always active and needed. They operate at a _______ level that is then induced or repressed as needed.
Basal
What type of prokaryotic protein represses gene expression by interacting with the gene promoter?
An oppressor protein
What type of prokaryotic protein increases gene expression by interacting with the gene promoter?
A gene activator
To what area of the gene do opressor proteins bind?
The repressor region
To what area of the gene do activator proteins bind?
Just adjacent to the promotor
What is typically the mechanism by which oppressor and activator proteins are activated or inhibited in prokaryotes?
Single effector signaling molecules
Regulation of the lactose operon depends on surrounding levels of what substances?
Lactose;
cAMP (glucose-dependent);
glucose
What is the signaling molecule for the lac operon?
cAMP
If cAMP levels are high, what happens to the lac operon?
It is activated
If lactose is absent, what happens to the lac operon?
A repressor binds it
If cAMP levels are low but lactose is present, what happens to the lac operon?
The lactose removes the repressor
cAMP binds what modifier to the lac operon?
The activator (CAP)
cAMP binds what modifier to the lac operon?
Lactose binds what modifier to the lac operon?
The activator (CAP) (inducing gene transcription);
the repressor (removing it and inducing gene transcription)
What effector binds the lac operon activator (CAP) and allows it to bind adjacent to the promoter, inducing transcription?
cAMP
What effector binds the lac operon repressor and allows it to unblock the promoter region, inducing transcription?
Lactose
What will be the state of transcription of the Lac operon in the following situation?
Lactose low
Glucose high
Low cAMP
No transcription
What will be the state of transcription of the Lac operon in the following situation?
Lactose high
Glucose high
Low cAMP
Some transcription
What will be the state of transcription of the Lac operon in the following situation?
Lactose high
Glucose low
high cAMP
High transcription
What scenario would lead to no lac operon transcription?
No lactose
Yes glucose
Low cAMP
What scenario would lead to a small amount of lac operon transcription?
Yes lactose
Yes glucose
Low cAMP
What scenario would lead to high lac operon transcriptional activity?
Yes lactose
No glucose
High cAMP
What is the eukaryotic equivalent of the prokaryotic promoter?
The proximal promoter
Where is the eukaryotic equivalent of the prokaryotic TATAAT sequence?
What is it called?
-30 BP;
TATA box
What eukaryotic protein binds the TATA box to organize the transcriptional process and identify the strand for usage?
TATA-binding protein (TBP)
How does most gene activation occur?
Which is more common, gene activating proteins or gene silencing proteins?
Heterochromatin to euchromatin changes;
activating (most silencing is simply the genome remaining as heterochromatin)
What is the name of the eukaryotic helicase that unwinds the DNA for transcription to take place?
What other activity does this enzyme have that allows it to activate the preinitiation complex and induce transcription?
TFIIH;
serine kinase activity
Phosphorylation primarily occurs on which histone’s tail?
Acetylation primarily occurs on which histone’s tail?
Methylation primarily occurs on which histone’s tail?
H2a
H2b;
H3
(remember PAM 2,2,3)
What is the important region of the prokaryotic promoter region to know?
If the gene of interest is +1, what is the region specified above?
TATAAT;
-10
Describe eukaryotic transcription initiation.
TFIIB and TATA-binding protein (TBP) bind what sequence and recruit what?
The TATA unit (-30);
the PIC (at the INR [-10])
What are some principal factors that make up part of the preinitiation complex of eukaryotic transcription?
RNA polymerase II,
TFIIH (activates RNA polymerase II),
TFIIE (activates TFIIH)
What part of RNA polymerase II is phosphorylated/activated by TFIIH and is also involved in hnRNA processing into mRNA?
The carboxy-terminal domain (CTD)
What two proteins of eukaryotic transcription bind the preinitiation complex (PIC) to begin the process?
The TATA-binding protein (TBP)
+ TFIIB
What is the name of the prokaryotic DNA sequence that is both adjacent to the gene of interest and also involved in controlling transcription of that gene?
What is the eukaryotic equivalent?
The promoter;
the proximal promoter
Where does the preinitiation complex (PIC) bind to the proximal promoter before initiating transcription?
The INR sequence (-10)
How can repressing proteins block eukaryotic transcription?
Inhibiting: the TATA-binding protein (TBP),
the preinitiation complex (PIC),
recruiting: histone deacetylation complexes (HDACs)
The template strand is also known as the ________ strand or the ________ strand.
noncoding;
antisense
(Templates Are Nonsense)
The noncoding strand is also known as the ________ strand or the ________ strand.
antisense;
template
(Templates Are Nonsense)
The antisense strand is also known as the ________ strand or the ________ strand.
template;
noncoding
(Templates Are Nonsense)
The TATA box is how many base pairs prior to the gene of interest (+1)?
And the initation region (INR)?
- 30;
- 10
Phosphorylation primarily occurs on which histone’s tail?
H2a
Acetylation primarily occurs on which histone’s tail?
H2b
Methylation primarily occurs on which histone’s tail?
H3
What is the generic name given to the complex that binds activators, the preinitiation complex (PIC), and RNA Polymerase II together at the beginning of transcription?
The mediator
What are some of the structures bound together by the mediator complex as transcription is being set up?
Activators,
the preinitiation complex (PIC),
and RNA Polymerase II
The mediator is required for proper PIC assembly because it binds:
Activators, the PIC and RNA Pol II
How does DNA polymerase detect and correct errors made during DNA replication?
3’ to 5’ exonuclease activity
True/False.
DNA replication in humans requires a free 3’ –OH group from a DNA primer.
False.
(it is an RNA primer)
True/False.
DNA synthesis is 5’to 3’ and replication moves from 5’ to 3’ on the template strand as well.
False.
(replication moves from 3’ to 5’ on the template strand)
True/False.
RNA primers are used in DNA synthesis in vivo and in vitro.
True.
Western blots probe what?
Northern blots probe what?
Southern blots probe what?
Protein;
RNA;
DNA
Which blot is useful for probing RNA?
Which blot is useful for probing protein?
Which blot is useful for probing DNA?
Northern;
Western;
Southern
When initiating transcription, what enzymes are actively ensuring the gene is open and available?
What enzymes are inactivated at this time?
HATs;
HDACs
What protein is responsible for the bending of DNA and the gene sequence in question into hairpin turn when transcription is being initiated?
HMG1
The mediator connects what structures during DNA transcription initiation?
The transcription factors/activators, TBP/TFIIB, and the PIC
What transcription factor is responsible for recruiting the transcription helicase (TFIIH)?
What factor identifies and binds the TATA box?
What transcription factor is responsible for phosphorylating the CTD of RNA polymerase II and also unwinding the DNA strands?
TFIIE;
TATA-binding protein (TBP);
TFIIH
