Unit 15 - DNA Damage and Repair

What are DNA damage the consequence of?

inaccuracy in DNA replication, chemical damage to the genetic material, and transposable elements

What does DNA damage lead to?

mutations

What can mutations lead to?

changes in coding or regulatory sequences, prevent use of DNA for replication or transcription, chromosome rearrangements

What are mutations?

permanent changes to the DNA

What is DNA damage?

something that is wrong at specific sequence in the DNA which can be repaired

What are point mutations?

base-pair substitutions

What are the two types of point mutations?

transitions or transversions

What is a transition point mutation?

change from purine to purine or change from pyrimidine to pyrimidine

What are other types of DNA mutations?

deletions, insertions, translocations

What is slippage?

change in the number of repeats of simple repetitive sequences such as CA

What does slippage during DNA replication produce?

an expansion or reduction in the number of repeats

How can the slippage lead to disorders?

can remain silent until the repeats build up enough and result in neurological disorders at older ages

What is responsible for the huntington's disease?

expansion of the CAG repeat in the huntingtin gene

What are the four challenges of repairing DNA damage?

taking place fast enough before permanent effect, distinguishing between the parental DNA and daughter DNA, response to cell DNA that blocks replication, and responding to double stranded breaks

What are the origins of mutations?

inaccuracy in DNA replication and chemical damage to genetic material

what is a mismatch?

transient, produced by failures of proofreading and are eliminated after a second round of replication

What test can we use to test mutagenic chemicals?

ames test

describe the ames test

the bacteria is mutated to require histidine, added to two separate plates both with no histidine, add mutagenic chemical to one plate and nothing to the other, see the growth

What would growth after adding the chemical mean?

confirms mutagenic properties as it cause the mutations to be reversed

What type of mutations are originally used in the ames test?

point mutations

What are the limitations of the ames test?

limited to prokaryotic cells, lack of metabolic activation in bacteria, and limited detection of certain mutation types

What does spontaneous hydrolytic damage of bases cause?

"unnatural" bases

What are the unnatural bases that are produced?

uracil, apurinic deoxyribose, and deamination of 5-methylcytosine

What are some common types of modification to guanine?

oxidation, alkylation, and deamination

Explain the oxidation of the guanine

oxidation of guanine produced OxoG which mispairs with adenine, causes the G:C to mutate to T:A

Explain an example of the alkylation of guanine

alkylation of O at C-6 produces O6-methylguanine which mispairs with thymine, G:C mutates to A:T

What is the most common mutations in human cancers and what is the origin?

OxoG, oxidizing agents and ionizing radiation

What is the thymine dimer formed by?

ultraviolet light

What is the thymine dimer?

photochemical fusion of adjacent thymine, which create covalent bonds

What are the impacts of the thymine dimer?

do not base pair and stop the DNA polymerase

What are base analogs?

chemical mutagens that can be incorporated into DNA, and then proceed to base-pair incorrectly, which introduces mutations into the DNA.

What are DNA intercalating agents?

flat molecules that intercalate between purine and pyrimidine bases

What are the impacts of the intercalating agents?

create distortions in the DNA template during replication, leads to insertions and deletions of bases

How are replication errors fixed?

mismatch repair pathway

Explain the mismatch pathway in E. coli

MutS scans the DNA rapidly and identifies mismatches by distortions in the DNA backbone, MutL and MutH bind, MutH will knick the DNA and use exonuclease to remove the fragment, which will allow DNA polymerase to fix the sequence

What helicase is utilized during the base mismatch pathway?

UvrD helicase

How does the MutH recognize the misplaced nucleotide?

methyl-directed mismatch repair and directionality in mismatch repair

Describe the methyl-directed mismatch repair

Dam methylase methylates adenine residues in sequence 5'-GATC-3', if not methylated it will nick the DNA there

How is the mismatch repair pathway performed in eukaryotes

dam methylase is not present, so MSH will interact with the PCNA and recognize the newly replicate strand using nicks in Okazaki fragment, uses pre-existing nicks

What is direct repair of DNA damage consist of?

photoreactivation and removal of methyl groups

What does photoreactivation repair?

pyrimidine/thymine dimers

How does the photoreactivation work?

DNA photolyase will use the energy from sunlight to directly reverse the DNA damage by break the bond between the thymine bases

What is the process of the removal of methyl groups from DNA bases?

methyltransferase will have a sulfur group which will attach the methyl group, removing it from the nucleotide

What are the two types of altered base replacement?

base excision repair and nucleotide excision repair

What happens during the base excisio repair pathway?

the incorrectly paired base will be removed from the DNA backbone

What is the enzyme that performs the base excision repair pathway

DNA glycosylase

What does DNA glycosylase do?

base-flips the DNA to check the accuracy of the nucleotide

What property of DNA makes the base-flipping possible?

flexibility of DNA

true or false: glycosylase are lesion specific

true

Describe the base excision pathway that occurs in the uracil glycosylase reaction

the glycosylase will remove the uracil, which will leave an abasic sugar and AP site, the AP endonuclease and exonuclease will cleave the segment of DNA from the DNA backbone

What groove of DNA do the glycolases diffuse laterally?

minor groove

Describe Oxo-G:A repair after translation

OxoG will be repaired by a fail-safe glycosylase that is dedicated to the recognition of Oxo-G:A pairs, does not impact the G rather exchanges the A for a C

What type of proteins are used in the nucleotide excision repair pathway?

prokaryotes: Uvr

humans: XP

What type of DNA damage do Uvr proteins repair DNA from?

UV light

What is special about the nucleotide excision repair pathway?

does not recognize specific lesion, rather recognizes the distortions in the double helix

Explain the process of nucleotide excision repair in prokaryotes

UvrAB is delivered to the DNA using ATP, UvrB opens the Dna and UvrA leaves, UvrC binds ad cleaves the DNA around the lesion, DNA helicase UvrD will open the DNA strands to allow DNA polymerase and ligase to repair the strand

XPC is equivalent to

UvrA

XPA, D is equivalent to

UvrB

XPG is equivalent to

UvrC

Why are individuals with XP mutations sensitive to light and frequently develop skin cancer?

the body is not able to perform nucleotide excision repair pathway and cannot fix the mutations in the genome

What is transcription-coupled repair?

repair that focuses on DNA that is actively transcribed

During transcription-coupled repair, what acts as the DNA damage sensing protein?

RNA polymerase

What makes the transcription-coupled repair possible?

XPA and XPD are components of TFIIH which allow the nucleotide excision repair proteins fix when RNA polymerase senses damage

What process occurs through damage tolerance?

translesion DNA synthesis

What is translesion DNA synthesis?

DNA synthesis that occurs even when DNA damage is present

How does translesion DNA synthesis occur?

DNA polymerase III and sliding clamp leave and Pol IV or V bind, which have larger active sites and will create base-pairs to the damaged lesion, DNA pol III and sliding clamp rebind after passing lesion

How can translesion DNA synthesis lead to mutations?

the Pol IV or V can place the incorrect nucleotides across from the lesion

What unrepaired DNA damage causes more damage than any other?

double stranded breaks

What can double stranded breaks lead to?

stalled replication and chromosome loss

What is the mechanism to repair DSB after replication?

homologous recombination based on the sister chromatid which is used as template for the repair

What is the mechanism of repair for DSB before replication?

non-homologous end joining

Describe the non-homologous end joining pathway

Ku70/80 will wrap around the end of the strand on both sides as this protein recognizes the break, preventing degradation, DNA-PKcs will bind with them, endonuclease artermis is found on the bottom of both ends of the kinase, which may trim back the DNA ends to create a clean end that can be joined together, DNA ligase IV enzyme, which associates with XRCC4 and XLF, will ligate the broken DNA ends together

why is the NHEJ a highly mutagenic process?

endonuclease and exonuclease will cleave nucleotides to create the clean end, increasing the probability of mutations