Chapter 25: DNA Metabolism Flashcards
TRUE/FALSE:
DNA is stable and not static
True
DNA metabolism includes the
processes of ____, _____, and ____
replication, repair, and recombination.
Proteins (sometimes Eukaryotic proteins) are usually written in Roman type and the first letter is capitalized. Example?
IspA, IspB, IspC, IspD, etc. (isoprene biosynthesis)
Bacterial genes (sometimes Eukaryotic genes) are written in lowercase italics, generally using three letters.
isp (isoprene biosynthesis)
If several bacterial genes are important for the same process, a fourth letter is added and it is capitalized.
Example?
ispA, ispB, ispC, ispD
DNA replication is_______ since each strand can act as a template for the synthesis of a perfect copy of the other strand.
semiconservative
What are the enzymes responsible for DNA replication?
DNA Polymerase
DNA Polymerase utilizes single stranded DNA
templates and adds nucleotides in what direction?
5’ to 3’
DNA molecule consists of one _____ and
one ____ .
old strand, new strand
replication “bubbles”
separation of two strands of DNA accompanied by synthesis of their complementary strands.
Origin of replication (oriC), a unique point, is where?
Replication always begins
Branch point in a replication bubble
Replication Fork
Most replication forks appear to be in what direction?
Bidirectional
New DNA base added to the ____ of the previously
added base
3’ OH
Semidiscontinuous Replication Model:
- Leading strand, is continuously
synthesized in the 5’ to 3’ direction. - Lagging strand, is synthesized in
the 5’ to 3’ direction in a discontinuous manner. - DNA ligase is required to join the discontinuous Okazaki fragments together into a continuous piece of DNA.
Because both strands are REPLICATED
simultaneously _____
both strands cannot be SYNTHESIZED continuously
What 8 proteins are required in replication?
- Nucleases
- DNA topoisomerases
- Helicases to separate DNA at the replication fork.
- Proteins to prevent reannealing
- Primases to synthesize RNA primers
- DNA polymerase
- An enzyme to remove the RNA primers
- Ligase to link together Okazaki fragments
What nucleases remove DNA only from the
ENDS of DNA strands?
Exonucleases
Most exonucleases operate in WHAT direction(s) ?
5’→3’ or the 3’→5’
(Most polymerases have a 3’→5’ exonuclease
activity)
What nucleases degrade DNA from the interior of a DNA strand (hydrolyze the phosphodiester bond)?
Endonucleases
Most endonucleases cut DNA internally with what enzymes?
restriction enzymes
DNA Polymerase:
Synthesize _____
Adds the incoming ____ to the 3’ OH of the growing
chain, releasing ___ in the process.
• This is a favorable reaction due to the subsequent hydrolysis of PPi and the additional base stacking and base pairing interactions that
occur.
• DNA polymerase requires a template that it can copy; it cannot
synthesize random sequences of DNA on its own.
• It also requires a primer with a free 3’-hydroxyl to which it can add
additional nucleotide bases. This primer generates a short
sequence of double stranded nucleotide. Most primers are actually
short stretches of RNA.
• After adding a nucleotide, the DNA polymerase can either move
along the strand and add the next base, or it can dissociate from the
strand.
DNA.
deoxyribonucleotide, PPi
What’s the name for dNTP?
Deoxynucleoside Triphosphate
DNA polymerase error rate is every
10^4 or 10^5
E. coli error rate is every
10^9 or 10^10
Proofreading and increases accuracy by 102- to
remove incorrect bases
principle replication enzyme
DNA Polymerase III
responsible for a variety of clean up functions
during replication, recombination, and repair
DNA Polymerase II
DNA polymerase II, IV, and V
involved in DNA repair
DNA Polymerase I contains ___ exonuclease activity
5’→3’
Which DNA Polymerase removes a short stretch of RNA or DNA and replaces it with a new sequence of DNA (nick translation)?
DNA Polymerase I
What enzyme creates RNA primers on the Okazaki fragments in E. coli?
Primase
DNA polymerase III requires a _____ for
addition of the next nucleotide,
free 3’ hydroxyl
Okazaki fragments’ 5’ ends consist of what?
RNA segments
In Prokaryotes, what DNA gyrase introduces negative supercoils (unwinding) into DNA replication, allowing the process to proceed
topoisomerase
What accessory proteins are needed to help DNA Pol III unwind?
DnaB helicase
Single-strand binding protein (SSB)
Can DNA Pol III unwind DNA?
No.
A hexameric ring is found in?
DNA Helicases
DnaB cycles between what three conformations?
NTP bound, hydrolysis, and release of products.
Single-Strand Binding Protein
Single stranded DNA (ssDNA) likes to anneal to form
dsDNA
DNA ligase is responsible for
ligating two pieces of DNA together to form a contiguous strand.
Energy for a DNA Ligase reaction is supplied by
either ATP hydrolysis to AMP + PPi or NAD+ hydrolysis to NMN+ + AMP
What are E. Coli replication steps?
initiation, elongation, and termination
Initiation takes place where?
Origin of replication (oriC)
Origin of replication (oriC) region denatures what and where?
which is an easily denatured 245 bp AT rich region
10 proteins involved in initiation?
- DnaA
- DnaB (Helicase)
- DnaC protein
- DnaG protein (Primase)
- HU
- FIS
- IHF
- Single-Strand DNA binding protein
- DNA gyrase (topoisomerase II)
- DAM methylase
Binding of an ATP-bound DnaA to the
five 9 bp repeats in the origin. ….complex denatures DNA in the three 13 bp DUE sequences
also located in the oriC.
The DnaC protein then loads DnaB onto the unwound segment of DNA.
The DnaB hexamers act as helicases and begin to unwind the DNA in both directions, creating the replication fork.
E. Coli Replication
The oriC DNA is methylated on N6 of adenine residues in the GATC sequences by Dam methylase. 11 of these sequences occur at the origin.
E. Coli Regulation
E. Coli Elongation requires synthesis of what?
synthesis of the leading strand and
the lagging strand
DNA helicases (DnaB) are required to unwind the
DNA, with the resulting stress relieved by
topoisomerases.
SSB binds to the single strands.
Primase (DnaG) then lays down short (10-60 nucleotide)
RNA primers at the origin.
DNA Pol III begins adding deoxyribonucleotides in a
continuous fashion to create the leading strand. … interacts with DnaB and travels along the replication fork
E. Coli Elongation
Synthesis of the next Okazaki fragment:
Okazaki fragment reaches the previous fragment, a
new clamp loads onto the next primer and associates with the lagging strand polymerase.
Replisome
the complex of proteins located at the replication fork
When an Okazaki fragment
is completed, what occurs?
1) . DNA Pol I removes the RNA primer and replaces it with DNA.
2) . DNA ligase then seals the “nick” to make a contiguous piece of DNA.
The two replication forks make their
way around the E. coli circular genome,
eventually meeting on the other side.
Multiple copies of a 20 bp termination
sequence (Ter) are found at the
opposite end; they trap the first
replication fork to arrive.
The other replication fork stops when it
meets the first one and the few hundred
bases between the forks are replicated
by an unknown mechanism.
Two circular chromosomes are interlinked
(catenated). Topoisomerase IV
separates the two chromosomes and
they are segregated into daughter cells.
E. Coli Termination
TRUE/FALSE:
Eukaryotic Replication is more complex than E. Coli and involves linear chromosomes.
True
TRUE/FALSE:
Eukaryotic AND Prokaryotic replication have the same basic mechanism
True
Mutations
are permanent changes in the genomic nucleotide sequence
What can cause Mutations?
Base changes or the Addition/Deletion of base pairs
Silent mutations
Have no effect on gene function
Most mutations are what type of mutation?
Silent Mutations
Though essential, repair systems are _____ and use ____ __ ____.
inefficient, lots of energy
Why is DNA repair possible?
Opposite strand will (hopefully) encode the
correct sequence.
TRUE/FALSE:
Mismatch Repair is a expensive process since 1000
bp or more may be hydrolyzed to repair a single mismatch.
True
When is the Mut Repair System required?
In prokaryotes, repair at sites distant from replication
MutL and MutS proteins form a
complex and bind to the mismatch site
(except C-C mismatches).
MutH then binds to MutL and the three
proteins thread the DNA through the
complex.
Once a hemimethylated GATC sequence is
encountered, the unmethylated strand is
cut by MutH, marking it as the strand for
repair.
DNA is unwound to the site of
mismatch and the new strand is degraded.
DNA is re-synthesized to
generate a proper DNA copy.
Mismatch Repair
Mismatch Repair corrects DNA using the
sequence of the ___ ___.
template strand
In Mismatch Repair, we monitor methylation of what residues?
Adenine
Homologs of MutS
bind to DNA as dimers
Homologs of MutS
MSH2, MSH3, and MSH6
Homologs of MutL
stabilize MSH complexes
Homologs of MutL
MLH1 and PMS1
TRUE/FALSE:
Defects in DNA repair proteins lead to increased susceptibility to cancer and other
diseases.
True
TRUE/FALSE:
Methylation of GATC residues is not used
to identify newly synthesized DNA strands.
True
Which homologs have been identified in humans and not well known?
MutH
DNA lesions (modifications of a base) are fixed by
base-excision repair
DNA glycosylase removes damaged base by cleaving Nglycosyl bond.
The deoxyribose 5’-phosphate left behind (apurine or
apyrimidine site) is removed by
an AP endonuclease.
Removal often takes out a segment of DNA.
DNA polymerase I replaces
the removed DNA before DNA
ligase seals the remaining “nick”.
Base-Excision Repair
When is Nucleotide Excision Repair used?
repair large distortions in the helical structure of DNA
In Nucleotide Excision Repair, multisubunit enzyme ___ ____ (includes the Uvr proteins A, B,
C, and D) hydrolyzes two
phosphodiester bonds, one on
either side of the distortion.
ABC excinuclease
What type of repair occurs when damage is repaired without having to remove the damaged base?
Direct Repair
When excessive DNA damage occurs, what type of repair in bacteria?
SOS Repair System
Which DNA Polymerase is activated in SOS Repair System?
DNA polymerase (pol V)
Though essential to the survival of the bacterial population, the SOS response leads to ___ __ ___
high error rates
What disease is associated with nucleotide excision repair?
Xeroderma pigmentosum (XP)
:extremely light sensitive because
they are unable to repair pyrimidine dimers formed by
UV light absorption.
What mismatch repair genes are associated with Hereditary nonpolyposis colon cancer?
MLH1 and MSH2
What mutations account for 10% of known breast cancer cases?
BRCA1 and BRCA2 (involved in DNA maintenance and
repair)
