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Flashcards in DNA Repair and recombination Deck (47):

How often do mutations occur in human genomes during replicaiton?

roughly 1 nucleotide per 10^8


What processes are able to correct errors in DNA replication?

- proofreading mechanism
- post replication repair


Low fidelity and high mutation rates occurring within germ cells results in what?

low fidelity and high mutation rates in germ cells causes cell death


Low fidelity and high mutation rates in somatic cells results in what?

Both will result in the formation and progression of cancerous cells


In order for DNA polymerase to function properly what materials does it require?

- dATP, dGTP, dCTP, dTTP
- strands separated by DNA helicase
- primers on 3'OH ends


What is the clamp loader protein?

- this protein hydrolyzes ATP in order to attach the sliding clamp onto the primer-template junction


Explain how DNA mismatch repair occurs in humans.

Lagging Strand:
- sequence is found via broken segments,
-MutS finds the mutation, MutL finds the closest "nick" and the DNA section between the two is removed and repaired.

Leading Strand:
- unknown process


DNA mismatch repair in E.Coli is flagged by what process?

The parental strand is methylated and signals that the other strand is the one that needs to be replaced.


What is the process that DNA Topoisomerase Type 1 operates?

- covalently bonds to one strand, cuts the other strand and allows the DNA to uncoil about the bound phosphodiester bond. Reduces coiled tension


How does topoisomerase 2 operate to relieve coiled tension?

-Used when two double helix cross paths.
--It cuts and bind both strands of one helix to itself, undergoes a conformational change to pass the other helix between the cut, and then rejoins the helix that was doubly cut.


Why is the repilcation origin often at sites with AT binding?

- AT only bond with two bonds, and is easily broken compared to GC.


What consists of tightly packed DNA that is most often replicated late?

- heterochromatin, the tight packing makes the replication process proceed slowly


Which DNA is loosely packed and is replicated faster and first?

-euchromatin is loosely packed


What are the minimum requirements for an active Origin of Replication?

1. binding site for Origin recognition complex
2. AT rich zone
3. Binding site for origin recogniction complex attraction protein.


What is a method to alter epigenetic inheritance?

-the recycling of histones from parental units, can retain some of the original patterning units and therefore pass on genetic material


What is the purpose of telomerase?

The telomerase is an enzyme that adds a specific sequence to the end of the DNA sequence so the lagging strand is fully synthesized. It places a repeating sequence on the end that allows the binding of a primer and DNA polymerase.


What are two syndromes that can result inf cancer due to defects in DNA repair mechanisms?

BRCA1: breast and ovarian cancer
BRCA2: breast, ovarian, prostate cancer


What is Werner syndrome and what causes it?

- premature aging, cancer, and gene instability
- caused by mutation in DNA Helicase repair


What is Bloom Syndrome?

- characterized by stunted growth, cancer, and gene instability
- caused by mutation in DNA helicase used in recombination


What are the most common types of spontaneous DNA damage?

1. depurination: removal of base from nucleotide
2. deamination: removal of N from the base
3. dimerization: UV radiation causes pyrimidine dimer


What types of repair processes can the cell use to fix damaged DNA?

1. base excision repair
2. nucleotide excision repair
3. transcription coupled repair


Explain how base excision repair works, and when it is most commonly used.

1.used with depurination mutations with the AP endonuclease.

2. DNA glycosylase recognizes and removes the glycosyl bond
- endonuclease and phosphodiesterase finish cleaving the rest of the strand to remove the damage.
- AP endonuclease replaces damage with proper sequence


How does nucleotide excision repair work?

1. an entire section of DNA strand is remoed, and is best for pyrimidine dimers, or chemically induced mutations.
2. DNA helicase, polymerase, and ligase are required in order to cut, unwind, and repair the damaged section.


Why is transcription-coupled repair useful to a cell?

- repair machinery is paired with transcription equipment. This helps RNA polymerase, if it encounters damage to the DNA it is reading. The repair machinery will repair the lesion, probably with mutation, but transcription will continue


Why in the humans is it acceptable to use transcription-coupled repair if there is a high likelihood it introduces mutation?

-only 1.5% of the genome is expressed as extrons, the remainder can be mutated to some extent and is better to have those mutations than it is to not have transcription taking place.


What is Cockayne's Syndrome?

- growth retardation, skeletal abnormalities, and UV sensitivity.

- caused by defective transcription coupled RNA polymerase


Why is RNA most likely to not be the genetic material?

-cysteine can undergo deamination causing it to appear as a uracil. If RNA were used there would be no way to distinguish between a mutated base pair of Uracil or a normal non-mutation


Deamination of a methylated C produces what?

thymine, which will create a lesion as G will be paired with T


Translesion Polymerase?

this polymerase functions just as DNA polymerase, however does not have a proofreading feature. Therefore it can transcribe lesions/mutation to DNA and allow transcription to continue with DNA Polymerase


How can double strand breaks be repaired?

1. non-homologous end joining
2. homologous recombination


What is non-homologous end joining?

1. major repair in Humans, and broken ends are ligated with DNA ligase


What are the processes that homologous recombination can be used to fix?

1. repair double stranded break
2. exchange and form new genetic sequences
3. mechanically ensure chromosome segregation


How does homologous recombination repair a double stranded break?

1. exonuclease degrade the 5' end of both strands
2. the 3' end invades the other newly formed DNA strand, matches and elongates the damaged strand to fill in the gaps.
3. the 'damaged' strand now becomes the template to fix the other s-strand sequence.
4. once done the new strands are ligated together.

this provides a very accurate way to repair


How can homologous recombination contribute to DNA hybridization?

1. Rec A/Rad51 bind with ssDNA and helps find a complementary sequence strand on the duplex DNA
2. ssDNA invades and inserts itself into the duplex DNA forming a heteroduplex DNA
3. the complementary sequence must be 15 bp long in order to attach.


Homologous recombination can be used to help polymerase continue when it runs into a broken section on the parental DNA strand. How?

1. 5' exonuclease trims back the sequence to prepare it for strand invasion
2. the overhanging 3' end invades the ds, synthesizes the required material, and detaches
3. the leading strand is acting somewhat like a lagging strand
4. the DNA polyermase is attached and transcription continues


What are the 4 general features of non-homologous end joining?

1. no template required
2. repairs site mutations
3. can create translocations
4. preferred method in human DNA


What are unique features of homologous recombination?

1. uses daughter DNA duplex as a template
2. no mutations/alterations occur at repair sites
3. can repair other DNA types of damage


What is a holliday junction?

DNA intermediate during crossing over that contains 4 strands of DNA from 2 separate helices, which is quickly cleaved via RuvC in a process called resolution


What is resolution?

DNA strand cleavage by endonuclease RuvC, during crossing over events or gene conversion


Which event, crossing over or gene conversion, is most commonly seen in humans?

-gene conversion at 90%


Both DNA strand of a Holliday junction are cut in the same direction. What is the final result?

1. gene conversion with minimal amounts of gene exchange


If both DNA strands are cut in opposite directions, what is the final result?

1. the upstream portion and the downstream portions are able to swap, resulting in crossing over.


When does gene conversion most likely occur and what is the primary outcome of this process?

1. occurs during heteroduplex mismatch repair.
2. causes loss of one allele and duplication of others.


What type of transposons are predominate in bacteria>?

DNA-only. allows sections of of DNA sequence to move to new sections.
- ability to spread AB resistance.
- the insertion site is duplicated, so if the sequence is excised, the genome will retain the original insertion site


In DNA-only transposons 3 different scenarios can take place. What is the cause of each of these scenarios?
1. restore transposon
2. unrestored transposon
3. mutation inserted

1. transposons are restored with ds break repair
2. transposons are unrestored with homologous recombination
3. mutations are inserted with nonhomologous end joining


What is unique about retrovirus-like transposons?

they function under the same premise as retroviruses but lack a capsid and are therefore unable to migrate to new cells.


Review how nonretroviral transposons, conservative site specific recombination, and site-specific recombination occur

Lecture 5 DNA processes 3