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Molecular Biology Dr. Ryals Exam 3
DNA replication and repair
78
Biochemistry
Undergraduate 4
03/29/2011

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Cards

Term
DNA replication must be these three thing
Definition

high fidelity (but need mutations)

highly processive

relatively fast

Term
Semiconservative replication
Definition

both strands get a new stand

this is the correct method

Term
conservative replication
Definition
both strands stay connected and a new one is created 
Term
dispersive replication
Definition
random pieces are kept and others are replaced 
Term
Meselson and Stahl experiment 
Definition

-demonstradted semiconservative

-applied CsCl gradient ultracenterfugation of DNA labeled with Heavy Nitrogen (15N)

-cells were moved to growth medium containing normal Nitrogen (14N)

-DNA was isolated at different times

-pictures taken during centerfugation

-found old strand of heavy DNA with new strand of light DNA

Term
continuous replicaiton
Definition

both strands serve as templates in the same compass direction 

one would do 3'-->5' and hte other would do 5'-->3'

Term
semidiscontinuous replication
Definition
same as continuous, but broken up into bursts on the same double strand 
Term
discontinous replication
Definition
fragmented on both parent strands 
Term
Okazaki proposed
Definition

-proposed that DNA polymerase can make one strand of DNA continously 5'-->3' (leading strand)

-but the other strand would be discontinous in 5'-->3' (lagging strand)

Term
Okazaki's model had two experimentally testable predictions
Definition

-if short pieces of DNA are synthesized on the lagging strand, we should be able to catch them with radiolabeling

 

-if the enzyme DNA ligase is eliminated from the replication process, then the short pieces of DNA made ought to be detectable even at longer labeling periods

Term
Okazaki's experiment
Definition

-used T4 phage mutant that made Ligase

-control - found short pieces for short periods of time before they were ligased together

-using the T4 mutant - tons of short pieces as time incresaed 

Term
RNA primers
Definition

required for DNA synthesis to begin

laid down on the template strands to provide a double-stranded region for DNA polymerase to bind

Term
DNA replication is _____ with DNA synthesis occuring ____ at ____ _____ ____
Definition

bidirectional

simultaneously

2 replication forks

Term
specific location where replication starts
Definition

oriC

contains 4 9-mers having a consensus sequence of TTATCCACA

Term
Cairns
Definition

-discovered the theta mode

-labeled replication E. coli DNA with radioactive DNA precursor and observed with autoradiography 

-did not display bidirectional replication

Term
Gyurasits and Wake
Definition

-radiolabeled replicating DNA for a short time with low levels of radioactive precursors, followed by high levels of radioactive precursor

-the nucleotides began to be incorporated in the new strands of DNA created

Term
Rolling Circle Replication
Definition

-not the theta mode

-example of unidrectional

-occurs in phage φX174 and i'm guessing  λ phage


Term
phage φX174 replication
Definition

-one strand of dsDNA is nicked and 3' end is extended

-intact strand is used as template

-a single stranded circle of DNA is released as single stranded circle of DNA

-normally exists as ssDNA but has to be double stranded for replicative form

Term
 λ phage replication
Definition

-strand nicked and 3' end laid out

-its this nicked strand that serves as template for discontinous lagging strand sythesis 

Term
helicase
Definition

-an ATP-dependent enzyme that separates the DNA strands in advance of the replication fork

-the dnaB gene product in E.coli

Term
Single-Stranded DNA-Binding Proteins
Definition

bind to ssDNA and prevent it from reforming dsDNA

-product of ssb gene of E.coli

-the bonding is cooperative - raises affinity by 1,000 fold for next molecule

-stimulate replication

Term
Topoisomerase
Definition

as the DNA unwinds, it has to wind somewhere upstream, creates strain

-topoisomerase releaves this strain by introducing temporary single- (Type I) or double-stranded (Type II) breaks in the DNA

Term
DNA gyrase
Definition

from E. coli

type II topoisomerase

Term
DNA Polymerases found in E. coli
Definition

Polymerase I - DNA repair, primer excision

Polymerase II - SOS repair

Polymerase III - required for DNA replication in E.coli

Term

Holoenzyme polymerase III subunits

aka: pol III holoenzyme

Definition

composed of multiple subunits

 

o   The α-subunit has the DNA polymerase activity

o   The ε-subunit has the 3’ à 5’ exonuclease proofreading activity

o   The θ-subunit has the ???? activity. The function is still unknown

 

Term
eukaryotic DNA polymerases
Definition

o   DNA polymerase α (priming)

o   DNA polymerase  δ (elongation)

o   DNA polymerase β (repair)

o   DNA polymerase ε (repair)

o   DNA polymerase γ (mitochondrial)

Term
DNA replication can be divided into 3 major events
Definition

inition

elongation

termination

Term
initation of replication - purpose of dnaA
Definition

-binds at the oriC

-facilitates the binding of dnaB

-stimulates melting of the 3 13-mer repeats at one end of the oriC to make an opening 

Term
initiation of replication - purpose of dnaB
Definition

-required for protein synthesis

-dnaC binds to dnaB

-stilumates binding of the primase

-also serves as the helicase that moves 5'-->3' on the lagging strand in the direction of the replication fork

Term
initiation of replication - two other factors for open complex formation at oriC
Definition

-RNA polymerase which synthesizes a short piece of RNA that creates an R loop

-helix unwinding (HU) protein which induces bending

Term
Initiation of replication - primase
Definition

-product of the dnaG

-its the RNA primer-synthesizing enzyme 

Term
initiation of replication - Primase (dnaG) + dnaB =
Definition

primosome 

-this is responsible for laying down multiple primers for Okazaki fragments on the lagging strand 

 

Term
how eukaryotes handle initiation
Definition

-multiple sites of replication for each chromosome

-and that's about all we know

Term
elongation - why pol III holoenzyme is highly processive
Definition

due to the sliding clamp

-its the β-subunit of the holoenzyme

-literally holds the entire pol III assembly on the template for long periods

Term
elongation - group of protein required for the sliding clamp
Definition

the γ-complex

-sliding clamp cannot touch the DNA by itself

-serves as the clamp loader and is ATP-dependent

Term
elongation - eukaryotes version of the sliding clamp
Definition

processivity factor PCNA

PCNA = proliferating cell nuclear antigen

-forms a trimer (3 subunits) that can encircle the DNA as the bacterial clamp does

Term
elongation - pol III holoenzyme
Definition

-contains two core polymerases - one for each strand

-as it finishes one Okazaki fragment, it runs into a nick that is positioned in front of the primer on the next fragment 

- this nick is a cue for the complex to dissociate from the and move to the primer on the next fragment 

Term
termination - the _____ region
Definition

terminus

-where the replication forks begin to get near each other

-contains 22bp sites that bind specific proteins called TUS proteins

-replication forks stop moving when they get to this region

-leaves the daughter duples entangled 

Term
termination - TUS proteins
Definition
terminus utilization substance 
Term
termination - when circular DNAs are interlocked (entangled), the structure is called ________
Definition
a catenane
Term
termination - Decantenation
Definition

-process of untangling the interlocking DNA rings

-preformed by topoisomerase IV

 

Term
termination - eukaryotes' problems
Definition

-there are gaps left when RNA primers are removed

-this is problematic because when the primer is removed DNA cannot be extended in the 3'-->5' direction

- and there's no 3' upstream

-so DNA should be getting shorter...

Term
termination - Elizabeth Blackburn
Definition

worked with Tetrahymena

discovered telomers

Term
terminiation - telomers 
Definition

-repeats of GC-rich sequences at the ends of eukaryotic chromosomes

-telomerase adds these strands, not DNA polymerase 

-exact sequence of the telomere is species specific and is also determined by the small bit of RNA inside the telomerase

Term
termination - telomerase
Definition

-carries a small RNA as part of its structure and the RNA serves as the template for telomere synthesis

-it is a ribonucleoprotein

Term

terminaton - which is which

TTGGGG

TTAGGG

Definition

TTGGGG - Tetrahymena

TTAGGG - vertebrates

Term
termination - Hayflick Limit
Definition

-Hayflick (1960s) showed that normal animal cell lines are not immortal

-grow in cultures of about 50 generations, then enter senescence

-cancer cells do not have this limit (contain telomerase as do egg and sperm production cells)

Term
DNA damage v. mutation
Definition

-not the same thing

-damage is a chemical alteration

-mutation is a change in a base pair

-unrepaired damage can lead to mutations

Term
Types of damages
Definition

-alkylation of bases

-UV-induced Cross Linking 

-Ionizing radiation

Term
Damage - Alkylation of bases - occurance
Definition
-when electrophiles bind to centers of negative change and add a carbon-containing group (alkyl group)
Term
damage - alkylation of bases - targets for attack
Definition

-phosphate groups

-N7 of guanine

-N3 of adenine

-O6 of quanine but is rare

Term
damage - alkylation of bases - alkylation of N7 of guanine
Definition
doesn't change the base pairing properties of guanine and is typically harmless lol 
Term
damage - alkylation of bases - alkylation of N3 of adenine
Definition

-creates 3-methyl adenine (3mA) that cannot base pair properly with any other base

-the 3mA is a noncoding base

-DNA polymerase stalls at it - doesn't compute 

-polymerase can go on and skip, but can result in erroneous replication which leads to mutations

Term
damage - alkylation of bases - O6 of guanine
Definition

-seldom but very mutagenic when it does

-the product can base pair with T instead of C

-occurs from the introduction of ethylmethane sulfonate

Term
damage - UV - dimers
Definition

-pyrimidine dimers by cross linking adjecent pryimidines on the same DNA strand 

-and is pretty common

-and they block DNA replication

-pretty much 2 Ts melt together so they look like one... and only 1 A is given to them

Term
damage - UV - cyclobutane ring
Definition

the 2 bonds that link the thymines form a four-memebered cyclobutane ring

and that's all i have 

Term
damage - ionizing radiation - caused by
Definition

-high energy x-rays and gamma radiation

-also called high energy radiation

-primarily caused by free radicals

Term
damage - ionizing radiation - free radicals
Definition

-form when water is ionized by the incident radiation

-have an unpaired electron and are extremely reactive

-especially the ones that contain O

Term
damage - ionizing radiation - clastogen
Definition

-breaks dsDNA

-which this does

-difficult to repair 

Term
DNA repair - two approaches
Definition

-directly repairing the damage

-removing the damage and replacing it with new DNA

Term
repair - undoing the damage in E. coli
Definition
photoreactivation and suicide enzyme repair
Term
repair - E.coli - photoreactivation
Definition

-light repair

-not in placental mammals but in all others...

-carried out by DNA Photolyase

Term
repair - photoreactivation - DNA photolyase
Definition

-recognizes and binds to the pryimidine dimers

-is activated when it absorvs light energy in the UV-A/blue (>300nm) part of the spectrum

-breaks the dimer bonds to reform monomer

Term
repair - E.coli - Suicide repair
Definition

-for the alkylation damage of O6 guanine

- also called O6-methylguanine methyltransferase

 

Term
repair - suicide - how it works
Definition

-the sulfur atom of a cysteine in the enzyme accepts the methyl or ethyl alkyl group

-this irreversibly inactivates the enzyme

-transferase is induced by DNA alkylation

Term
repair - excission and replacement - mechanisms
Definition

base excission repair (BER)

nucleotide excission repair

Term
repair - BER - enzymes required
Definition

DNA Glycosylase

AP Endonuclease

DNA Phosphodiesterase

DNA Polymerase I

DNA Ligase

Term
repair - BER - DNA Glycosylase
Definition
breaks bond between damaged base and its sugar
Term
repair - BER - AP Endonuclease
Definition
nicks the DNA on the 5' side of the AP site
Term
repair - BER - DNA Polymerase I
Definition
degrades DNA in the 5'-->3' direction while filling in with new DNA
Term
repair - BER - DNA Phosphodiesterase
Definition
removes the AP sugar phosphate
Term
repair - BER - DNA Ligase
Definition
seals the nick
Term
repair - what AP stands for
Definition

Apyrimic or Apurinic

means without the bit it is missing lol 

Term
repair - NER - forms
Definition

Global Genome NER

Transcription-Coupled NER

Term
repair - NER - transcription-coupled NER
Definition

-confined or restricted to transcribed parts of the genome

-only occurs in parts of the genome being actively transcribed

-RNA polymerase will stall at the lesion leaving a region of melted DNA

Term
repair - NER - global genome NER
Definition

-will work on any DNA within the genome

- repair to any lesion damage throughout the genome can occur

Term
repair - global genome repair - XP
Definition

-Xerroderma Pigentosum

-thousands of times more likely to get skin cancer

-helix distorting damage such as dimmer formation goes unrepaired due to the defective NER

Term
repair - global genome repair - how XPC-HhR23B works
Definition

-recognizes the distortion in the helix and binds to it

-induces localized DNA melting around damage

-RPA binds to the undamaged DNA across from it

-TFIIH, a helicase, causes additional melting

-RPA helps position 2 endonucleases (ERCCI-XPF complex and XPG) on either side

-an oglionucleotide containing the damage is excised

-DNA polymerase ε or δ fills the gap

-ligase seals the nick

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