Flashcards in DNA Metabolism Deck (100)
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1
Exonuclease
Nucleases that remove DNA only fron the ENDS of DNA strands.
-Operate only in the 5'->3' or 3'->5' direction
-Hydrolyze Pohophodiester bonds (FROM THE END)
-ALL E.coli DNA polymerases have 3'-5' exonuclease activity (proofreading capability)
-E.coli DNA polymerase I also have 5'->3'
2
Endonuclease
-Degrade DNA from the INTERIOR of DNA strand
(Hydrolyzes the phosphodiester bond)
-Cut internally only at specified sequences (high specificity to where they cut)
-A.K.A. Restriction Enzymes
3
DNA Polymerase
-Synthesize DNA
-Adds incoming deoxyribobucleotide to the 3' OH of the growing chain, releasing PPi in the process
(This is a FAVORABLE reaction due to the subsequent hydrolysis of PPi and the additional base stacking, base pairing interactions)
-After adding Nucleotide, DNA polymerase can move along the strand and add the next base or it can dissociate from the strand
4
Template
DNA polymerase requires it to copy
5
Primer
DNA polymerase requires it with a free 3' hydroxyl to which it can add additional nucleotide bases.
-Generates a short sequence of double stranded nucleotide.
-Most primers are short stretches of RNA
6
Processitivity
Some DNA polymerases add thousands of nucleotides before dissociating meaning they are extremely processive
7
E-coli has an error rate of...
10^9 or 10^10 bases incorporated
-Ecoli Chromosome is about 4.6 x10^6 bp so a mistake is made every 1,000-10,000 replications
8
The corret dNTP is determined by...
-Shape
-Hydrogen Bonding
as base pairs with the incorrect geometry (shape) will not fit into the active site (Non binding makes Kd go way up!)
9
DNA Polymerase makes a mistake for every...
10^4 or 10^5 incorporated bases.
10
Polymerase Proofreading
3'->5' exonuclease activity- used to remove incorrect bases and increase accuracy by 10^2 to 10^3 fold
-Conformation-change driven (evolution!)
11
E.coli has at least _____ different DNA polymerases, each of which has a specific function.
5
12
DNA Polymerase III
The principle replication enzyme
-3'->5' Exonuclease
>10 Subunits
13
DNA Polymerase I
Responsible for a variety of clean up functions during replication, recombination, repair
-3' to 5' Exonuclease
-5'->3' Exonuclease
-1 Subunit
14
DNA Polymerase II, IV, and V
Involved in DNA repair
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DNA Polymerase I rate/Processivity
Polymerization Rate (Nucleotides)(Also Kcat Turnover #): 16-20
Processivity (nucleotides added before polymerase dissociates): 3-200
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DNA Polymerase II Subunits/ rate/Processivity
Subunits: 7
Polymerization Rate (Nucleotides)(Also Kcat Turnover #): 40
Processivity (nucleotides added before polymerase dissociates): 1,500
17
DNA Polymerase III rate/Processivity
Polymerization Rate (Nucleotides)(Also Kcat Turnover #): 250-1,000
Processivity (nucleotides added before polymerase dissociates): >500,000
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DNA Polymerase I
*Seperate subunit which contains 5'-3' exonuclease activity
-Cleans up Okazaki Fragments
-This allows Pol I to be involved in NICK TRANSLATION
-Lays down new DNA Strand
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Nick Translation
Removal of short stretch of RNA or DNA and replaces it with a new sequence of DNA
-Important for many repair mechanisms and replacement of RNA primers
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DNA Polymerase III
-Ecoli's DNA REPLICASE.
-Enzyme is similar to Polymerase I but is part of a complex of at least 13 subunits of 9 different types
Theres a...
-core polymerase
-Claw- clamp loading complex working with beta subunit
-Clamp
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If DNA Polymerase III require a 3' OH for addition of the next nucleotide, where does the first hydroxyl come from?
RNA
22
RNA Primase
Creates RNA primers on the Okazaki Fragment in E.coli
-These primers are eventually removed from mature DNA and filled in by DNA Polymerase I (because of its 5'-3' exonuclease)
23
Topoisomerase
(AKA DNA GYRASE)
-If you unwind DNA as replication proceeds, you will eventually reach a point where the DNA becomes so tightly coiled that it can no longer be unwound.
-E. Coli replication becomes tightly coiled (because they replicate at a rate of 1000 base pairs/second)
-Prokayotes use DNA Gyrase to induce negative supercoils into DNA, allowing the process to proceed
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What does DNA Pol III use to unwind DNA?
-DNAB Helicase
-Single Stranded Bining Protein (SSB)
*DNA B unwinds DNA and once unwound, SSB will bind to it to keep it from reannealing
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DNA Helicases
-Form a Hexameric ring
-Moves along the DNA strans while hydrolyzing NTP
(traverses 5' to 3' but only on ONE Strand)
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DNAB cycles between 3 conformations:
-NTP Bound
-Hydrolysis
-Release of products
*Conformational changes unwind the dsDNA
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Single-Stand Binding Protein
Single stranded DNA (ssDNA) likes to anneal to form dsDNA
-Done to keep the recently unwound DNA from reassociating
-E.Coli SSB is a tetrameric protein that coats ssDNA
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DNA Ligase
Responsible for ligating 2 pieces of DNA together to form a continuous strand
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Energy supplied for DNA Ligase comes from...
-ATP hydrolysis to AMP +PPi
-NAD+ Hydrolysis to NMN+ + AMP
-An Adenylate moiety is added to the 5' phosphoryl terminus to promore ligation
*Example of adenlyation reaction of enzyme AND substrate
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E.coli replication can be divoded into steps of...
-Initiation (takes place at the origin of replication oriC- easily denatured AT rich region)
-Elongation
-Termination
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Proteins involved in initiation (7)
-DnaA:
-DnaB
-DnaC
-Primase (DNA G)
-Single stranded DNA binding protein (SSB)
-DNA Gyrase (DNA Topoisomerase II)
-Dam Dethylase
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Proteins that stimulate Initiation (3)
-HU
-FIS
-IHF
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-DnaA:
Recognizes Ori Sequence- opens duplex at specific sites in origin
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-DnaB
unwinds DNA
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-DnaC
Required for DNAB binding at origin
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-Primase (DNA G)
Synthesizes RNA primers
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-Single stranded DNA binding protein (SSB)
Binds Single stranded DNA
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-DNA Gyrase (DNA Topoisomerase II)
Relieves torsional strain generated by DNA unwinding
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-Dam Dethylase
Methylates (5') GATC sequence at OriC
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E.Coli Replication Initiation Step 1
Step 1: binding of an ATP-bound DnaA to the five 9 bp repeats in the origin. This complex then denatures the DNA in the three 13 bp DUE sequences located in the ori C as well.
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ATP bound DNA A is....
ACTIVE
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ADP bound DNA A is...
NOT ACTIVE
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DNA A is a slow...
ATPase
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ADP bound DNA A can interact with...
I sites and R sites (this is what initiates replication)
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How does DNA A recognize those 5 bp sequences at the origin?
There are base specific interactions in the major groove. Alpha helix sits in the major groove of DNA.
46
DnaA: Alpha Helix has 5 side chains, what happens if a base in the stretch is changed?
Kd goes UP (low affinity) because base specific interactions change. If Kd is low, then it is going to saturate genome at this site.
***Kd dictates OriC***
47
DnaA: Major Groove interactions with backbone phosphate groups.
-Interaction with phosphate groups does NOT give specificity
-Contributing Interactions: Non-Covalent bonds, NOT covalent because protein would be anchored for ever
-Enhance the interaction via electrostatic interactions
-Kd is LOWER (energetics are affected)
-LAH will like to interact (electrostatic interactions)
48
DnaA: Minor Groove interactions (eventhough alpha helix doesnt fit in the minor groove)
-proteins reacts and sticks "fingers: in the minor groove to anchor it.
-Kd is turned DOWN, ensuring Dna A binds to Ori C where its supposed to
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E.Coli Replication Initiation Step 2
-DnaC protein (chaperone) loads DnaB onto the unwound segment of DNA
-DnaB hexamers act as helicases and begin to unwind the DNA in both directions, creating the replication fork
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E.Coli Replication Initiation Step 3
ATp bound Dna C begins opening up hexamer and then lets the single strand go
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E.Coli Replication Initiation Step 4
DnaC hydrolysis of ATP causes release of DnaC from DnaB
-unwinding DNA as it travels and driven by ATP hydrolysis- ATP bound, ADP + Pi, bound, mt
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E.Coli Replication Initiation Step 5
DNA B Hexamer acts as helicase as DNA unwinds in both directions (forming replication fork)
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E.coli Initiation Regulation
Regulation of DNA replication is poorly understood
54
Dam Methylase
During Inititation Regulation:
Methylates OriC DNA on N6 of Adenine residues in the GATC Sequence
-11 of these sequences occur at the origin
55
E.coli Initiation Regulation: Step 1
-Dam Methylase Methylates
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E.coli Initiation Regulation Step 2
Immediately after replication, the origin is HEMImethylated (only 1 strand is methylated, preventing replication from begining) and sequestered at the plasma membrane
57
E.coli Initiation Regulation Step 3
Full Methylation by Dam Methylase is required to initiate replication again.
58
What dictates initiation of replication?
Proper methylation is regulating when initiation begins as well as DNA A (whether its bound to ATP or not)
59
E.Coli Elongation Players
Elongation requires synthesis of the loading strand and lagging strand.
-DNA Helicases (including 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. It interacts with DnaB and travels along the replication fork.
60
SSB
-Binding site of single stranded binding proteins is lined with hydrophobic aromatic groups.
-4 amino acids are important for single stranded binding: TRP40, TRP 54, TRP88, PHE60
-bind sites will stack with the bases
61
In Elongation, how does beta clamp associate with DNA/RNA hybrid? How does it stay there?
They generate an aritifical RNA/ DNA hybrid
-Put on a chloraform to see it easier with rystals
-see DNA located on inside of clamp
62
E.coli Termination Step 1
-The 2 replication forks make their way around the E.Coli circular genome, eventually meeting on the other side
63
E.coli Termination Step 2
Multiple Copies of a 20 bp termination sequence (Ter) are found at the opposite end; they trap the first replication fork to arrive
64
E.coli Termination Step 3
The other replication fork stops when it meets the first one and the few hundred
65
E.coli Termination Step 4
At this point, the 2 circular chromosomes are interlinked (catenated). Topoisomerase IV seperated the 2 chromosmes and they are segregated into daughter cells
66
Replication Organization
2 replication forks are tethered together at the bacterial inner membrane and DNA feeds trough this repliaction factory
-2 completed genomes are partitioned into seperate halves of the dividing cell.
(Pic)
67
Eukaryotic Replication
Replication is slower thank in prokaryotes, occuring at 1/20 of that in E.Coli, only about 50 nucleotides/second.
-more complex, involving linear chromosomes
-requires origins of replication (replicators)
-Replication proceeds from many origins-making it copy chromosomes in a reasonable time
68
Mutations
Permanent changes in the genomic nucleotide sequence and can be due to base changes or the addition/deletion of base pairs
-Can be SILENT because they do not have an effect on gene function
69
Ames Test
Rate of mutations
70
Carcinogen
Mutates at position that produces cancer
71
Do repair systems for DNA exist?
Yes, a variety because DNA gets damaged, tend to be inefficient and use alot of energy. Possible because hopefully, opposite strand will encode the correct sequence.
72
4 Repair Systems are...
-Mismatch Repair
-Nucleotide-Excision Repair
-Base-Excision Repair
-Direct Repair
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Mismatch Repair
-Repairs rare mismatches incorporated by DNA polymerase
-Corrects DNA using the sequence of the template strand- so it has to determine NEW and OLD DNA
74
Mismatch Repair: How E.coli distinguishes strands (old vs.. new)
-*Monitors Methylation of Adenine Residues*within GATC sequence by Dam methylase.
-The original strand is methylated while it usually takes a few minutes after replication to methylate the new strand.
(Parent Strand-Methylated, Daughter Strand-NOT)
75
Mismatch Repair: mismatches can be repaired up to...
1,000 bp from a hemimethylated GATC sequence.
76
Mut Repair System
Repair at sites distant from replication require Mut Repair Systems in PROKARYOTES
77
MutL and MutS Functions
These proteins form a complex and bind to the mismatch site (EXCEPT C-C mismatches)
78
MutH and MutL bind
and Bind with 3 protein thread the DNA through Complex
79
Mismatch repair: How does an enzyme recognize mismatch?
MutS determines the Mismatch
80
Mismatch repair: How does MutS determine there's a mismatch?
Residue interactions and interactions with the mismatch site (mismatch bonds dont hydrogen bond well)
-Anti-conformations of nitrogenous bases is favored in duplex DNA.
-MutS has PHE that pushes against the base pairs
-The ones that are correct stay in their configuration and the ones that are mismatched are released because they are not energetically favorable
-Glu is ready to catch any base that are about to swing from anti->syn configuration (AND THIS IS HOW MUTS RECOGNIZES MISMATCH)
-Kd Drops
-MutS is forced to serve as a nucleation site for the rest of the process
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Eukaryotic Mismatch Repair: Homologs of MutS
MSH2, MSH3, MSH6-appear to bind to DNA as dimers
82
Eukaryotic Mismatch Repair: Homologs of MutL
MSH1,PMS1-stabalize MSH complexes
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Eukaryotic Mismatch Repair: Is Methylation of GATC residues used to identify newly synthesized EUKARYOTES?
NO, Methylation of GATC residues is not used to identify newly synthesized DNA Strands.
84
Eukaryotic Mismatch Repair: What happens when theres a defect in DNA repair proteins such as MutS homologs?
leads to increased susceptibility to cancer and other diseases
85
What does Base Excision Repair fix?
Common DNA lesions
86
How does Base Excision Repair work?
First, the modified base is removed by DNA glycosylases which cleave the N-glycosyl bond to generate an apurinic (AP) or apyrimidic (abasic) site
87
Base Excision Repair: What does DNA glycosylase do?
Removes damaged base by cleaving N-glycosyl bond
88
Base Excision Repair: What does AP endonuclease do?
Removes the Deoxyribose 5'-phosphate left behind (apurine or apyrimide site)
-this removal often takes out a segment of DNA- can take bigger stretches of DNA.
89
Base Excision Repair: What does DNA polymerase I replace?
Replaces the removed DNA before DNA ligase seals the remaining nick
90
Nucleotide Excision Repair:
Used to repair large distortions in the helical structure of DNA (which can be caused by UV damage)
91
Nucleotide Excision Repair: ABC Exinuclease
-Includes the UVR proteins A,B,C,D
-hydrolyzes two phosphodiester bonds, one on either side of the distortion.
92
Direct Repair:
Sometimes the damage can be directly repaired without having to remove the damaged base:
-Often happens when an unwanted methylation occurs, thoughout replication- base pairs change and properate mutation.
-For example in O6-Methylguanine- which can lead to G-C, A-T mutations
93
Direct Repair: What can unwanted methylation cause?
Can cause G to pair with T and through replication, keeo passing on daughter strand to subsequent generations (this is the mutation)
94
Direct Repair: Methyl Transferase function
Enzyme that accepts the methyl group from O6-methylguanine and generates an inactivated enzyme.
-Body sacrifices this enzyme to get rid of methyl group.
-Enzyme has an active site Cysteine which becomes methylated
-Enzyme is sacrifices for the DNA to make sure proper genetic information is copied.
95
SOS Repair:
-Bacteria using SOS response are destined to be dead so do whatever they can doto stay alive longer
-Caused by excessive DNA damage
-Leads to higher error rates
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SOS Repair: Polymerase V (Pol V)
Can replicate past many DNA lesions and activates under SOS conditions
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Xeroderma pigmentosum (XP)
Results from the defect in nucleotide excision repair
-People are extremely light sensitive bc they are unable to repair pyrimidine dimers formed by IV light absorption
-Mutations in 7 different proteins give rise to XP
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Heredity nonpolyposis colon cancer
-Linked to defects in mismatch repair genes (MLH1, MLH2)
-Cancer develops early
-
99
BRCA 1, BRCA2 Mutations
account for 10% of breast cancer- proteins involved in DNA maintenance/repair
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