Lecture 12: Chromatin Structure & DNA Replication Flashcards
(119 cards)
1
Q
A diploid cell has how many base pairs of DNA?
A
About 6 billion
2
Q
DNA is organized in the nucleus as?
A
Chromatin
3
Q
Chromatin is a complex of what two things?
A
DNA and proteins.
4
Q
Chromatin is dynamic, it can…
A
compact or expand, depending upon the circumstances
5
Q
What is the organization component of DNA?
A
Histones
6
Q
Overall structure of connected nucleosomes resembles. They are called?
A
Beads on a string. Histones (histones are proteins)
7
Q
Nucleosomes
A
Histones and their associated DNA
8
Q
Nucleosome consists of? Shortens?
A
DNA wrapped around a core made up of different histones. This structure shortens the DNA length about 7 fold.
9
Q
In the nucleosome, _____________ 146 bp of DNA wraps ____________ times around a histone protein
A
146 bp of DNA wraps 1.75 times around a histone protein core
10
Q
Histone core consists of how many proteins?
A
- 2 molecules each of histones H2A, H2B, H3 and H4. The term nucleosome refers to the DNA and the 8 core histones.
11
Q
Chromatosome includes the
A
DNA, the 8 core histones, and histone H1, covering 165 bp.
12
Q
Histone H1 is involved in
A
stabilizing higher order chromatin structures (NOT part of the core)
13
Q
Histone tails are?
A
Modification sites.
14
Q
The tighter the histone-DNA complexes, the more?
A
Difficult access for replication or transcription becomes.
15
Q
Acetylation of the lysine residues (positively charged) in the histone reduces?
A
Interaction with the negatively charged DNA (more relaxed)
16
Q
Histone acetyltransferases (HATs)
A
add acetyl groups to the lysines in histone tails (more relaxed)
17
Q
Histone deacetylases (HDACs)
A
remove the acetyl groups
18
Q
HDAC inhibitors
A
Block HDAC function. Used in cancer treatment.
19
Q
Naked DNA
A
fully de-condensed, transcriptionally active
20
Q
Histones and chromatosomes are found in?
A
interphase chromatin
21
Q
Loop domains are anchored to?
A
Scaffold proteins
22
Q
Mitotic chromsomes
A
fully condensed, transcriptionally silent
23
Q
The structural organization of DNA regulates
A
gene expression. Proteins needed for transcription must be able to access the DNA.
24
Q
Euchromatin
A
When the cell is not actively dividing (during interphase), much of the DNA exists as the more de-condensed 10 and 30 nm fibers organized as loop domains (transcriptionally active).
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Heterochromatin
The remaining DNA exists as highly condensed form; transcriptionally inert
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Constitutive heterochromatin
DNA sequences that are not usually
transcribed (centromeres or telomeres). Compacted and gene-poor.
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Facultative heterochromatin
Genes that are not transcribed in that cell type,
but may be in other cell types.
-Example is the X-chromosome in
mammalian females. One of the two X chromosomes is packaged as heterochromatin. It does this to balance the dosage of X linked genes between males and females, only one of the two chromosomes in females cells becomes inactivated. Can be altered based on stages/cell needs, dynamic.
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Semiconservative replication
the process by which by which both strands of
a double-stranded piece of DNA are
copied
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Result of semiconservative replication
two double stranded pieces of DNA that are identical to the original generated
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The two strands of the parental double stranded DNA must be separated so that
complementary copies can be made of each strand
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Nucleoside
a nitrogenous base linked to a
sugar only (without phosphates)
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Nucleotide
a nitrogenous based linked to
a sugar and between to 1 – 3 phosphates
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The sugars are
ribose (RNA) and deoxyribose (DNA)
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Ribonucleoside triphosohates (NTPs, or ribonucleotides) are used to make?
RNA
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Deoxyribonucleoside triphosphates (dNTPs or deoxyribonucleotides) are used to make
DNA
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The N in NTP or dNTP can indicate any of the?
Bases (ATP or dATP, CTP or dCTP)
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Purines
Double rings. Adenine (A) and Guanine (G)
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Pyrimidines
Single ring. Cytosine (C), Uracil (U), Thymine (T)
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DNA and RNA both have
adenine, guanine, and cytosine
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Thymine is specific for? Uracil is specific for?
DNA and RNA
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RNA is
single stranded (mostly)
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What is responsible for the double stranded nature of DNA?
Hydrogen bonds between the bases of each DNA strand (dsDNA)
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Each base will only "pair" with?
Another base that has a complimentary arrangement of atoms to allow formation of hydrogen bonds
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What are the base pairs?
G-C (DNA and RNA)
A-T (DNA only)
A-U (RNA only)
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How many hydrogen bonds per each base pair?
G-C = 2
A-T or A-U = 2
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Hydrogen bonding is critical for?
Stabilization of dsDNA.
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More hydrogen bonds between base pairs =
more energy needed to separate that pair.
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The correct incorporation of deoxyribonucleotides into a growing DNA strand is primarily determined by?
Hydrogen bonding
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Hydrogen bonding is also critical for:
Transcription. Correct hydrogen bonding between single stranded DNA and unincorporated ribonucleotides facilitates addition of the correct bases to the growing RNA.
Note: The RNA strands do not remain bound to the DNA template
strand.
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Damage to a nucleotide that
changes its hydrogen bonding
characteristics will result in
the wrong nucleotide being incorporated in the new strand during DNA replication. A mutation!!
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When cytosine undergoes deamination, it becomes
(C U later)
Uracil. (Uracil binds adenine)
(C to G --> U to A)
(C U later)
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Deamination of adenine
becomes hypoxanthine. Binds Cytosine.
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Deamination is
spontaneous
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Deamination can create
mutant proteins
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The carbon positions of the ribose ring are designated with an apostrophe (’), which is referred to as
"prime," to distinguish these carbon positions from those of the base
ex: 2' = 2 prime
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Ribose contains an
–OH group on its 2’ carbon
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Deoxyribose lacks?
2'OH group
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1' carbon
attaches to the nitrogenous base
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2' carbon
-H (DNA) or -OH (RNA)
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3' carbon
site of new nucleotide addition during replication or transcription
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4’ carbon
anchors the 5’ carbon
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5’ carbon
attaches to the phosphate groups
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The DNA strands are
antiparallel
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What type of bond for backbone of DNA?
phosphodiester bond
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The 5' end has a free
phosphate
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The 3' end has a free
OH group
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DNA synthesis begins at
replication origins (specific DNA sequences)
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Origin-binding proteins ((origin replication complexes (ORCs))
recognize the origins and begin to separate the DNA strands, forming a replication bubble
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Replication proceeds
bidirectionally form each origin until the replication forks meet each other or the end of the chromosome is reached
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What is critical fore DNA elongation?
A free 3'-OH group
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Because a 3'OH group is always needed, the elongation of a new DNA strand can only proceed from?
5' to 3' end
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What attaches nucleotides to the 3' (OH) end of the DNA strand?
All known DNA polymerases
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DNA is built in the what direction? By creation of what bonds?
5' to 3' by the creation of phosphodiester bonds
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All DNA polymerases require a 3’-OH in order to covalently bond nucleotides
into the
growing daughter strand
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DNA pol a. Function.
RNA primer.
Has the ability to synthesize short stretches of RNA on a template without a 3’-OH.
The 3’-OH from this RNA primer serve as as the point for initiation of new DNA
synthesis
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The RNA primer is later replaced with the
corresponding DNA nucleotides
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DNA synthesis on both the leading and lagging strands begins with
DNA polymerase alpha a,
which is composed of multiple subunits that each have distinct activities (DNA primase subunit, DNA polymerase subunit)
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DNA primase subunit
A DNA-dependent RNA polymerase that synthesizes a short RNA primer (~10 nucleotides).
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DNA polymerase subunit
synthesizes a short stretch of complimentary DNA (~ 20 nucleotides)
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The bulk of DNA replication is then performed by two additional polymerases
1. DNA polymerase d
2. DNA polymerase e
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DNA polymerase d
synthesizes the Okazaki fragments of the lagging strand
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DNA polymerase e
synthesizes the new DNA on the
leading strand
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DNA primase/polymerase a first synthesizes
a single RNA/DNA primer (green)
82
DNA polymerase ε
Then takes over to continue extending the new DNA strand towards the replication fork.
DNA polymerase ε can do this continuously
as long as it has a single-stranded template
with which incoming nucleotides can base
pair.
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The lagging strand synthesizes new
DNA in the opposite direction of the replication fork
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On the lagging strand, what synthesizes multiple RNA/DNA primers?
DNA primase/polymerase a
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A new RNA/DNA primer is needed each time a new
Okazaki fragment is synthesized.
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Each Okazaki fragment is then elongated by
DNA polymerase d away from the replication fork until the new fragment meets a previous Okazaki fragment
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DNA primase/polymerase a then starts a new
RNA/DNA primer closer to the replication fork, as new single stranded DNA is revealed by the separation of the parental strands.
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RNase H
1. Recognizes the RNA portion of any RNA/DNA duplexes
2. Then removes the RNA primer, leaving the DNA part intact
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DNA polymerase d (Okazaki)
fills in the resulting
nucleotide gap with DNA
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Lagging strand is synthesized in what direction
3' to 5'
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DNA ligase
repairs the nicks in the phosphodiester backbone, joining the fragments together.
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DNA ligase can also fill in other
phosphodiester gaps in the new DNA
strand, such as those which occur as
the result of replication forks coming
together
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Additional proteins needed at
replication fork
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DNA helicase
ravels just ahead of the leading strand DNA polymerase and uses the energy of
ATP hydrolysis to break the hydrogen bonding between individual base pairs, separating the two strands.
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Single stranded DNA binding proteins (SSB proteins)
bind to exposed, single-stranded DNA
of both strands once the helicase has separated them.
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SSB proteins stabilizes the
single-stranded molecules, preventing re-association of the two strands and intramolecular hydrogen bonding. This has the effect of straightening small
stretches of the DNA molecule, facilitating replication.
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Helicase
causes the DNA ahead of the replication fork to twist, forming supercoils, which would eventually DNA synthesis
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Topoisomerase
enzymes which relieve the strain by causing nicks in the DNA that allow one or both strands to rotate relative to the other
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Two classes of topoisomerases
Topoisomerase I (nicks one strand)
Topoisomerase II (cuts both strands)
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Topoisomerase I covalently attaches where? Creates what?
Covalently attaches to a phosphate of the DNA backbone, creating a nick in only strand of the DNA
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The strands rotate relative to?
each other to help relieve the strain on DNA
102
The high energy bond of the topo-phosphate linkage does what?
Provides the energy for spontaneous reformation of the phosphodiester bond - which reseals the nick in the DNA (no ATP is required to reseal the nicked DNA)
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Topoisomerase II uses
ATP hydrolysis to create double stranded breaks in DNA
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Rather than cause a rotation in one strand, the creation of what allows what?
Creation of double-stranded break allows one segment of double-stranded DNA to pass through another.
105
Topoisomerase II Steps
1. Toposiomerase binds to double helix 2
2. ATP hydrolysis is used to create a break in Helix 2
3. The broken ends of Helix 2 are held by Topo II
4. Double helix 1 is now able to pass between the broken strands of Helix 2
5. Helix 1 is released
6. The broken ends of Helix 2 are resealed
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Telomeres
the terminal sequences of linear DNA molecules consists of repeats of simple sequence DNA
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Telomerase
telomeres are maintained by telomerase, which catalyzes the synthesis of telomeres in the absence of a DNA template
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The RNA template (in telomerase) allows for
telomerase to extend the 3' end by one repeat unit beyond its original length
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the complementary strand can then be synthesized by?
polymerase a-primase complex
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Bacterial express two different types of topoisomerase enzyme?
DNA Gyrase
Topoisomerase IV
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Fluroquinolones
a class of broad-spectrum antibacterial medications that inhibit both of these bacterial (but not human) topoisomerase enzymes, thereby affecting the ability of bacteria to synthesize new DNA
112
In the US, nine different fluroquinlones are currently approved for human use, including
ciprofloxacin (Cipro) and levofloxacin (Levaquin) and
moxifloxacin (Avelox).
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Fluoroquinolone drugs are some of the most
Commonly prescribed antibiotics in the U.S. They can have some serious side effects including tendon rupture (may interfere with collagen turnover).
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Many drugs used to treat viral infections are
called
“chain terminators” because they
effectively terminate the construction of a
new strand of nucleic acid.
115
Chain terminator drugs
nucleoside or nucleotide analogs. They are similar enough in structure that DNA polymerases will try to incorporate them into new nucleic acid strands during
replication.
116
Acyclovir
antiviral drug widely used to treat HSV-1 and HSV-2 infections. This drug is an analog of the ribonucleoside guanosine (guanine + ribose sugar). It can be incorporated into the growing
strand of DNA but LACKS 3’ OH group on
which to add another nucleotide.
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