The Mutability and Repair of DNA Flashcards
(32 cards)
What are some sources of mutations/damage to DNA?
- inaccuracy in DNA replication
- spontaneous damage
- exogenous damage: radiation, chemicals, by-products of cellular metabolism
What are 2 major consequences of DNA damage?
- mutations (gene or regulatory sequences)
- lesions or structural changes to the DNA can prevent its use as a template for replication or transcription
What are point mutations and what are the two types that exist?
They alter a single nucleotide 1. transitions: purine to purine or pyrimidine to pyrimidine
A - G or T - C
2. transversions: purine to pyrimidine or vice versa
What are 4 different mutations that can occur in coding regions?
- silent
- missense
- nonsense
- frameshift
- base addition
- base deletion
silent mutation
doesn’t change the amino acid
missense mutation
a change in the amino acid
nonsense mutation
stops protein synthesis because it turns into a stop codon
frameshift mutation
base is added or deleted to change the animo acid sequence from that point on
A major limit to DNA replication accuracy is___.
The occasional flickering of the bases into the wrong tautomeric form
amino –> imino
keto–> enol
The alternate tautomer for each base changes its base-pairing specificity. It allows the incorrect bp to be correctly positioned for catalysis during DNA replication and incorporated into the daughter DNA.
1 in 10^5 nts
DNA polymerase has _____ proofreading activity.
3’–> 5’ exonuclease activity
this is a separate active site on the same polypeptide as the polymerase active site; it detects and removes mistakenly added nucleotides.
decreases the error rate of DNA replication by a factor of 100
The 3 stages that define the accuracy of DNA replication.
- DNA polymerase incoporation “kinetic proofreading”
- 3’ exonuclease activity of DNA polymerase
- postreplication mismatch repair
Mismatch Repair
A second chance to repair replication errors that escape proofreading
What are the two challenges of mismatch repair?
- find mismatches in the genome quickly. They are transient and after a second round of replication the mismatch is eliminated and leads to a mutation that won’t be detected
- repair the mismatch accurately; choose the correct nucleotide to replace
How does Mismatch repair occur in E.coli?
- MutS (dimer) scans the dsDNA and detects mismatches based on the distortion to the DNA structure. This forms the MutS-DNA complex where MutS binding to a mismatched bp induces a kink in the DNA and a conformational change in the MutS protein.
- MutL is then recruited and activates MutH. MutH is an endonuclease that creates a single stranded nick in the DNA near the mismatch. It binds to the hemimethylated GATC sites following replication, but its endonucelase activity is only activated when MutS/MutL detects a mismatch nearby. Following activation, MutH selectively nicks the unmethylated DNA strand
- an Exonuclease digests the DNA from the nick past the mismatched nucleotide
- single strand gap is filled in by DNA polymerase and sealed by DNA ligase
How do you identify the parental template from the newly synthesized strand?
(in E.coli only)
Dam methylase: methylates “A” residues at 5’ GATC 3’ sequences on parental strands; hemimethylated
-newly synthesized daughter strands are initially unmethylated
T/F. Mismatch repair can require either 5’ or 3’ exonuclease activity.
true
How does mismatch repair work in Eukaryotic cells?
Eukaryotic mismatch repair machinery includes MutS, MutL homologs (MSH and MLH)
- mutations in MSH and MLH genes leads to a genetic predisposition to colon cancer
- no MutH homolog; hemimethlation is not used to tag the parental strands; okazaki fragments?
Hydrolysis
Spontaneous DNA damage
- unnatural bases/ alterations
1. spontaneous deamination of cytosine generates uracil
2. spontaneous depurination produces an abasic/ apurinic site in the DNA where the deoxyribose sugar lacks a purine base - natural base/alteration
3. deamination of 5-methylcytosine generates thymine. this will not be recognized as an abnormal base. It can lead to a transition mutation if unprepared
Radiation- UV light
thymine dimer is formed where it is incapable of base pairing and interferes with DNA replication and transcription
O6-methylguanine
DNA damage
methylation of guanine to O6-methylguanine will mispair with thymine. after additional DNA replication this will lead to a GC to AT transition mutation
Intercalating Agents
DNA damage
cause the deletion or addition of a base pair or a few; can lead to frameshift mutations
i.e- ethidium, proflavin, acridine orange
What is the most cytotoxic DNA damage that can occur?
Ionizing radiation like X-rays cause double stranded breaks in the DNA backbone!
What are two direct reversal mechanisms of DNA damage?
- photo reactivation: damaged bases are directly repaired: DNA photolyase breaks the covalent bonds linking adjacent pyrimidines like thymine dimers, using energy from light
- methyl group removal from O6-methylguanine
What is Base excision repair?
- DNA glycosylase removes damaged bases by hydrolyzing the glycosidic bond between a damaged base and its deoxyribose sugar. This generates an basic (OH) residue in the backbone. DNA glycosylases are specific. There are 11 in human cells.
- Abasic residue is excised and replaced. How? The basic residue gets excised from the DNA backbone via endonuclease/exonuclease activity, leaving both a free 3’OH group and a 5’phosphate. A repair DNA polymerase fills in the gap using the undamaged strand as template and DNA ligase seals the nick.
Endonucleolytic cleavage also removes abasic residues that arise from spontaneous hydrolysis like depurination of G residue
