Lecture 11 Flashcards
What are some environmental agents that damage DNA
Environmental agents such as UV light, ionizing radiation and some chemical agents can damage DNA.
What are mutations
alterations in the DNA structure that can produce permanent changes in the genetic information encoded if they are not repaired.
What mutation causes sickle cell anemia?
Glu6 -> Val change in hemoglobin
How can mutations be silent
if they are in non-essential regions of the DNA, or have a negligible effect on the gene
How can mutation in genes that encode proteins that control cell cycle and DNA repair can lead to cancer if not repaired?
For example, mutations could inactivate tumor suppressor proteins, which control cell growth, or could over-activate oncogene products that stimulate cell growth, with both types of mutations leading to uncontrolled cell growth. Mutations in genes that encode for DNA repair proteins means that natural errors are not repaired, increasing the chance of mutations in tumor suppressor genes and oncogenes
What are the two types of mutations
- Point mutations: substitution of one base pair for another because of base mismatches usually caused by modification/damage of bases
- Insertions/deletions of one or more base (“indels”) often generated by “DNA intercalating” agent
Which DNA pol has 3->5 proof reading fxns
1,2, and 3
How does base mismatch usually occur
when the template base has been modified (damaged) so that its H-bond donor/acceptor arrangement now favors a base other than the one it normally pairs with. If this error isn’t corrected, this strand will now direct an incorrect base to incorporate during DNA replication, when it is used as a template.
Two types of base substitution?
Transition and tranversion
What is transition?
The most common base modifications result in replacement of one purine (e.g., G for A) by another purine or of one pyrimidine by another pyrimidine (e.g., C for T).
- e.g., G becomes modified such that it now base pairs with the pyrimidine T instead of the pyrimidine C.
What is transversion?
Less frequently a purine is replaced by a pyrimidine or vice versa - called a tranversion
-e.g., G becomes modified such that it now base pairs with A instead of C – the pyrimidine C has been replaced with the purine A.
When happens when the modification (e.g., G ->G) is not quickly repaired by cellular DNA repair enzymes, and DNA pol I or III don’t catch the resulting mismatch (GT)
change will be introduced into the daughter strand and become permanent -> a mutation.
What mutations are caused by chemical mutagens that modify bases
- deamination (removes an amine group)
- alkylation (adds a methyl or ethyl group, usually to purines)
What are deanimating agents? Example?
- deaminates aromatic primary amines (induces a mutation from A-T to G-C)
e. g. nitrous acid
Treatment of DNA with nitrous acid results in deamination of adenine, what is the product?
Hypoxanthine
What does hypoxanthine base pair with and why?
base pairs with cytosine because it can form two of guanines’ three H-bonds with cytosine
How is nitrous acid formed
formed from organic precursors such as nitrites
Can deanimation occur spontaneously?
Cytosine can become deaminated to form uracil, resulting in a C-G -> to T-A transition
(C-G -> U-G -> U-A -> T-A)
How are deanimated bases repaired?
Base excision repair
What are three alkylating agents
- ethylnitrosourea
- dimethlysulfate
- dimethylnitrosamine (found in food preservatives)
Which reactive oxygen species produced by normal cellular metabolism can cause DNA alkylation?
superoxide ion .O2-, hydroxide radial .OH
What is O^6-alkylguanine (also called O^6- methylguanine or mG)
is highly mutagenic -
base-pairs with T instead of C (GC -> AT mutation)
What is O^6-alkylguanine alkyltransferase’s role
usually the modification/damage caused by O^6akylguanine is repaired before a mutation occurs – “direct repair” by the DNA repair enzyme O6-alkylguanine alkyltransferase, which transfers the methyl group to a cysteine in its active site (the enzyme becomes permanently alkylated)
What are chemical mutagens that cause insertions/deletions?
Intercalating agents
What do acridines do to the DNA double helix?
Flat aromatic molecules that can intercalate between adjacent base pairs in the DNA double helix. Their presence can lead to the insertion or deletion of one or more base pairs.
What is ethidium bromide?
a chemical used in the lab to visualize DNA on agarose gels intercalates between bases, becoming intensely fluorescent when exposed to UV light is a chemical mutagen
Most important source of DNA damage?
Oxidative damage by reactive oxygen species like hydrogen peroxide, hydroxyl radicals and superoxide radicals arise during irradiation or as a byproduct of aerobic metabolism
Which radical is responsible for most of the damage?
hydroxyl radicals, highly reactive – removes an e- from any molecule in its path, generating a new free radical
What do radicals and superoxide radicals do?
cause a range of damage including oxidation of deoxyribose and bases and strand breaks
What do ionizing radiation do to DNA
ionizing radiation (X-rays, gamma rays) can also cause ring openings, fragmentation of bases, breaks in the sugar-phosphate backbone of DNA (strand breaks)
How do cells convert reactive oxygen species to harmless products?
cells have an elaborate defense mechanism (eg. catalase, superoxide dismutase)
How do cells repair damage caused by reactive oxygen species?
cells also have elaborate DNA repair mechanisms to repair damage
What enzyme repairs damage from UV light and ionizing radition?
DNA glycosylases using base excision repair
How does UV light damage DNA?
- UV light induces the condensation of 2 ethylene groups (C6=C5) in adjacent thymines on the same DNA strand to form a cyclobutane ring
- adjacent thymines can also dimerize via a C6-C4 bond (6-4 photoproduct)
What effect do thymine dimers have on DNA?
block replication and transcription because the helix distortion (kink) blocks polymerization past this site
How to correct the “lesion” caused by thymine dimers?
this “lesion” can be corrected by a photo- reactivating enzyme photolyase that binds specifically to thymine dimers and uses visible light to hydrolyse the bonds linking the pyrimidine rings
the ability of an organism to survive UV irradiation directly correlates with what
with its ability to remove thymine dimers from its DNA
What is photo-reactivation
a method of direct repair of DNA - occurs in bacteria and lower eukaryotes (including plants)
What can unrepaired thymine dimers do?
Lead to melanoma in humans
Because human cells do not contain photolyases how are thymine dimers repaired?
by nucleotide excision and in some cases base excision repair
What is xeroderma pigmentosum (XP)?
Rare skin disease caused by mutations in a # of human nucleotide-excision repair genes
Repercussions of XP?
- people with XP are extremely sensitive to sunlight or ultraviolet light - die from melanoma and other skin malignancies (cancers)
- changes in the skin become evident in infancy and worsen with time - skin becomes dry, and there is a marked atrophy of the dermis
What suit is used to protect people with XP
A prototype of a UV-protection suit to help people suffering from the genetic disorder xeroderma pigmentosum. The suit consists of two items: a headgear protecting the head and face, and a suit covering the rest of the body, which together filter out 100% UV light from the sun.
What are the 5 ways to repair DNA?
- Direct repair
- Base excision repair
- Nucleotide excision
- Mismatch repair
- Post-replication repair
Describe direct repair? Examples?
damaged bases are not removed but repaired “on-site” e.g., O6-alkylguanine alkyltransferase, photolyase for thymine dimers
Describe base excision repair
offending base is removed and replaced eg. bases modified spontaneously or by chemical mutagens
Describe nucleotide excision repair? Example
offending nucleotide is removed and replaced, eg. thymine dimers in eukaryotes
Describe mismatch repair
when the wrong nucleotide is added during DNA replication – system must discriminate between the template (correct) and the newly synthesized strand (incorrect), many enzymes involved
Describe post-replication repair
important in bacteria, involves recombination
5 steps in base excision repair
Base excision repairs DNA when a base of a nucleotide is damaged, for example cytosine
- Cytosine can easily lose an amino group, forming a base called uracil
- Uracil cannot form a base pair with guanine
- An enzyme, glycosylase, discovers the defect and excises the base of uracil
- Another couple of enzymes remove the rest of the nucleotide from DNA strand
- DNA polymerase fills in the gap and the DNA strand is sealed by DNA ligase
Describe nucleotide excision repair in 4 steps
Nucleotide excision repairs DNA-injuries caused by UV radiation or carcinogenic substances like those found in cigarette smoke
- UV radiation can make two thymines bind to each other incorrectly
- The enzyme exinuclease finds the damage and cuts the DNA strand. 12 nucleotides are removed
- DNA polymerase fills in the resulting gag
- DNA ligase seals the DNA strand. Now the injury has been dealt with
Describe mismatch repair in 5 steps
When DNA is copied during cell division, mismatching nucleotides are sometimes incorporated into the new strand. Out of a thousand mistakes, mismatch repair fixes all but one
- Two enzymes, MutS and MutL, detect the mismatch in DNA
- The enzyme MutH recognizes methyl groups on DNA. Only the original strand, which acted as a template during the copying process, will have methyl groups attached to it.
- MutH cuts the unmethylated strand with mismatch (i.e newly synthesized strand)
- Exonuclease I degrades the DNA in the 3’->5’ direction through the mismatch
- DNA polymerase fills in the gap and DNA ligase seals the DNA
Nobel prize in chemistry 2015 was awarded for what
“For mechanics studies of DNA repair”
- mismatch repair
- nucleotide excision repair
base excision repair
