Lecture 19: DNA Replication and Repair Flashcards
DNA Synthesis
What does DNA polymerase require?
- Template (copy)
- RNA primer with 3’ OH
- dNTP substrates
DNA Synthesis
What does DNA polymerases catalyze?
-Nucleophilic attack by 3’ OH
- Phosphodiester bond
- 5’ → 3’ synthesis
DNA Synthesis
What is a SNUG active site?
A SNUG active site can only accommodate “correct” base pairs
DNA Synthesis
In what direction does DNA always occur?
DNA synthesis is always 5’ -> 3’
Possible Models For Replication
Describe Semiconservative replication?
Hybrid of old and new strands
Possible Models For Replication
Describe Conservative replication?
Duplex of only old OR only new
Possible Models For Replication
Describe Dispersive replication?
Alternative new/old strand pieces throughout duplex
- Complementarity allows replication with high fidelity
DNA Polymerases
What do DNA polymerases do?
Accurately and efficiently replicate the genome
- They join individual nucleotides to produce a new strand of DNA
DNA Polymerases
What does DNA Polymerase III do?
What is a advantageous quality does it have?
Replicates the genome
Highest processivity
DNA Polymerases
What is processivity?
The number of nucleotides incorporated by a polymerase in a single binding event
DNA Polymerases
What are the two types of exonuclease activity that occur in DNA replication?
3’ -> 5’ exonuclease activity
and
5’ -> 3’ exonuclease activity
DNA Polymerases
What does 3’ -> 5’ exonuclease activity involve?
Proofreading
- allows for the removal of one base at a time
DNA Polymerases
What does 5’ -> 3’ exonuclease activity involve?
Removing primers
- Allows for the removal of many bases at a time
DNA Polymerases
What direction do polymerases work in?
All have 3’ -> 5’ exonucleocase activity for proofreading
- This allows for removal of one base at a time
DNA Polymerases
How often is an incorrect nucleotide inserted?
By how much do DNA Polymerases improve accuracy?
- Inserted approx every 10^4 to 10^5 bps
- Improves accuracy 10-100x
DNA Replication
What does DNA Polymerase I do?
Has 5’ -> 3’ exonuclease activity
- Removing primers
- Allows for the removal of many bases at a time
DNA Replication
What are the three major steps in DNA replication?
1) Initiation
2) Elongation
3) Termination
DNA Replication
Where does DNA replication occur in Eukaryotic cells?
In the nucleus
DNA Replication: Initiation
What are the the 5 specific sequences and proteins involved in initiation of DNA replication?
1) DUE (DNA Unwinding Element)
2) DnaA
3) DnaB
4) DNA gyrase/ topoisomerase II
5) SSB
DNA Replication: Initiation
What does DUE do?
DUE = DNA Unwinding Element
- Makes AT rich easier to melt
- It can be unzipped with less energy
AT-rich region is exactly the site where a replication complex is formed and where the DNA synthesis is initiated
DNA Replication: Initiation
What does DnaA do?
recognizes oriC sequence (where
duplex splits)
DNA Replication: Initiation
What does DnaB do?
Helicase: Unwinds DNA
DNA Replication: Initiation
What does DNA gyrase/topoisomerase II do?
relieves strain generated by unwinding
DNA Replication: Initiation
What does SSB do?
Binds to single-strand DNA
- helps stabalize
DNA Replication: Initiation
What steps occur during Initiation?
1) DnaA binds at oriC
2) DUE region (part of origin) is
denatured
3) DnaC-dependent loading of DnaB helicase
DNA Replication: Elongation
What steps occur during Elongation (part I)?
● Starts at the origin of replication (circular DNA)
● Primase lays down RNA primers
○ At origin for leading strand
○ Before each Okazaki fragment in lagging strand
● DnaB helicase unwinds DNA
● DNA polymerase III adds nucleotides
● SSB stabilizes strands
● DNA gyrase relieves strain by unwinding
DNA Replication: Elongation
What is the difference between elongating the leading vs. lagging strand?
DNA polymerase III can only works from 5’ to 3’
Leading strand synthesis is continuous
Lagging strand synthesis is discontinuous
- Leads to formation of Okazaki fragments
- Primer is laid before each one
DNA Replication: Elongation
What does the beta clamp do?
Which strand uses the beta clamp the most?
Intended to make sure DNA polymerase does not fall off the DNA strands
- The lagging strand requires constant loading and unloading of beta clamp, which requires ATP hydrolysis
DNA Replication: Termination
What steps occur during termination?
What kind of DNA topoisomerase is involved in this process?
1) Replication forks stop in terminus region
2) After termination, the two duplicated chromosomes are catenated (entangled)
3) DNA topoisomerase IV seperates the two chromosomes
DNA Replication in Eukaryotes vs. Prokaryotes
Name the 3 major differences in DNA replication in Eukaryotes vs. Prokaryotes
Replication in Eukaryotes is…
1) A LOT slower and more complex
2) Chromosomes in eukaryotes are long and linear
3) Initiation requires 2 steps
DNA Replication in Eukaryotes vs. Prokaryotes
Initiation in Eukaryotes requires 2 steps, what are they?
1) Formation of a pre-RC (replication complex)
- CDK enzymes off (cyclin-dependent protein kinases)
2) Coordinate activation
- CDK enzymes
- Involves ATP hydrolysis
Telomeres
What are telomeres, what do they do?
- Repetitive nucleotide sequence at end of chromosome
- Shorten at end of each replication cycle
- Don’t care if we lose them (they don’t encode for anything!)
Telomeres
What problem do telomeres help fix?
Problem with ends of linear chromosomes
- Ends will not get replicated
Telomeres
What does Telomerase do?
Synthesizes DNA from RNA template (reverse transcriptase)
- Template is part of enzyme
Reverse transcription
What is reverse transcription?
RNA-dependent DNA synthesis
Reverse transcription
What are examples of Reverse Transcriptase?
1) HIV (retrovirus since it uses a reverse transcriptase)
3) LINE-1 (retrotransposon)
DNA Damage
What are genetic mutations?
a permanent change in the DNA sequence
DNA Damage
What are the different types of genetic mutations?
Describe them
Silent: No effect on Gene function
- No change in AA sequence
Deleterious: impaires gene function
Advantageous: enhances gene function
- Will be selected for and maintained
DNA Damage
What are the different types of base substitutions?
- Transitions
- Transversions
- Base insertion
- Base deletion
DNA Damage
Describe a transition base substitution
- What do they lead to?
A transition occurs when
- pur -> pur
- py -> py
Very frequently leads to synonymous changes in code (so do not change AA sequence in protein)
DNA Damage
Describe a transvertion base substitution
- What do they lead to?
A transvertion occurs when
- pur -> py or vice versa
Can significantly change protein sequence, less common
DNA Damage
What might cause a genetic mutation to occur?
- Deamination
- Depurination/Alkylation
- UV Radiation - Thymine Dimer
DNA Damage
What is deamination?
What specific deamination is frequent, what does this deamination lead to?
It is…
- Removal of amino acid group
- NH2 to a carbonyl
Frequent..
- Deamination of of cytosine is very frequent
- How you can get a Uracil in a DNA molecule
DNA Damage
What might cause deamination at high levels?
Deaminating agents can induce conversions at high levels
DNA Damage
What are examples of Deaminating agents
- Nitrosamine
-Sodium nitrate - Sodium nitrate (nitrous acid precurors)
DNA Damage
What is Depurination/Alkylation?
How does this happen, and why?
What is an example of this?
Alkylation can change base pairing properties
Happens spontaneously, due to alkylating agents
Example
- O6-methylguanine will
base pair with thymine instead of cytosine
DNA Damage
What is the most common of type of modification that UV radiation causes?
kink creates block to replication and transcription!
cross-linking (covalent bond) of cyclobutanes of neighboring pyrimidines: formation of thymine dimers
DNA Damage
What do people that have impaired repair of UV damage inspired DNA lesions develop?
They develop a disease called xeroderma pigmentosum
- extreme light sensitivity
DNA Repair
What are the 4 types of DNA Repair Mechanisms?
1) Mismatch
2) Base Excision
3) Nucleotide Excision
4) Direct Repair
DNA Repair
What type of errors does mismatch repair correct?
How does it correct these errors?
Corrects replication errors through methylation
DNA Repair
What is the mxn behind mismatch repair?
- MutL-MutS binds to mismatch
- MutL-MutS + MutH find Me site
- MutH cleaves unmodified strand
- Exonuclease activity degreades DNA from the Me past mismatch pair
- DNA pol II replaces the degraded DNA by copying methylated parent strand
DNA Repair
What type of damage does base excision repair?
What enzymes does this repair use?
Repairs damages bases
- example:uracil in DNA due to cytosine deamination
Uses different glycolysase for each base lesion
DNA Repair
What is the mxn behind base excision repair?
- DNA glycolysase cleaves N-glycosyl bond
- AP endonuclease removes sugar
- DNA pol I removes the nick and adds a new base
- DNA ligase seals nick
DNA Repair
What damage does Nucleotide-excision repair fix?
What enzyme does it employ?
Removes bulky lesions where more than one base is affected
- Excinuclease
DNA Repair
What is the mxn behind nucleotide-excision repair?
- Exinuclease makes two cuts around the affected area to remove damaged DNA
- DNA helicase removes the damaged DNA
- DNA pol I adds new bases
- DNA ligase seals new fragments with old DNA
DNA Repair
What does Direct Repair do?
Repairs defect directly
- do nor remove any bases or nucleotides
DNA Repair
What are the mxn of direct repair?
Metabolically expensive
- Uses a “suicide enzyme”
- Cost: one protein per repair (protein CANNOT be used again)
