DNA Repair, Recombination, and Rearrangement Flashcards
Things that cause damage to DNA
- ionizing (x/gamma rays) and UV radiation
- methylating reagants such as MNNG
- cross-linking reagents such as the anticancer drug cisplatin
- bulky hydrocarbons such as bento-a-pyrene (carcinogen from tobacco smoke)
DNA repair mechanisms
- photoeactivation
- removal of alkyl guanines
- nucleotide excision repair
- base excision repair
- mismatch repair
- double-strand break repair
- daughter strand gap repair
mutation
any heritable (permanent) change in the structure of an organism’s DNA or chromsomes
loss of function mutations
- gene product has less or no function
- phenotypes are most often recessive
- exception: haploinsufficiency
null allele
- allele that has a complete loss of function
- amorphic mutation
haploinsufficiency
when the reduced dosage of a normal gene product isn’t enough for a normal phenotype
gain of function mutation
- change the gene product such that it gains a new and abnormal function
- usually have dominant phenotypes
- aka neomorphic mutation
dominant negative mutation
- aka antimorphic mutation
- have an altered gene product that acts antagonistically to the normal allele
- usually result in altered molecular function (often inactive)
- characterized bu a dominant or semi-dominant phenotype
lethal mutation
- lead to a phenotype incapable of effective reproduction
- leads to death
whole chromosome or genome level mutation
- fragment deletions/insertions (gene aberrations)
- inversions
- ploidy changes
molecular or base-pair level mutation
- deletions
- insertions
- substitutions (point mutations)
origins of mutations
- errors during DNA replication
- errors in chromosome alignments & separation during mitosis and meiosis
- spontaneous chemical changes in base structure
- induced chemical changes in base structure
polymerase I & III function
- exonuclease proof-reading
- corrects mistakes
tautomeric shifts
- spontaneous mutations
- the spontaneous isomerization of a nitrogenous base to/from keno and enroll forms or to/from amino and amino forms
- cause transition mutations
- replacement occurs in second generation of replication
transition mutation
one purine/pyrimidine base pair is replaced with the other purine/pyrimidine base pair
transversion mutation
replacement of a purine//pyrimidine with a pyrimidine/purine pair
Sickle Cell Anemia
- effect of a single missense mutation
- B-hemoglobin
- mutation in hemoglobin causes Hb^s phenotype
- glutamic acid is instead mutated to valine (hydrophilic to hydrophobic amino acid)
- hydrophobic nature causes the hemoglobin to oligomerize
principal cause of cancer
genomic instability resulting from defective repair of DNA damage
Endogenous DNA-damaging reactions
- depurination of adenine
- deamination of cytosine
- oxidation of guanine
- methylation of guanine
- oxidation of thymine
depurination
removes purine from backbone of nucleotide
deamination
removes amine from part of the base and turn it into a different base
oxidation
occurs most often
-why you should eat antioxidants!!!
photolyases
- repair DNA damage
- use light energy to break bonds linking pyrimidine rings
- mammals and frogs don’t have this repair mechanism
- uses FADH to break pyrimidine-pyrimidine bonds
- 1 of 2 enzymes that uses direct repair
alkyltransferases
- repair DNA damage
- 1 of 2 enzymes that uses direct repair
- methylating agents yield modified bases (that could induce mutagenesis or death if not repaired)
- some used in cancer therapy bc of ability to block replication
- most common product of alkylation = O6 methyl guanine (can lead to AT mutations if not repaired)
- alkyltransferase can only function once, but alkylated form can activate the transcription of the gene encoding alkyltransferase
nucleotide excision repair
- indirect mechanism
- used to repair thymine dimers and bulky adducts
- involves bending DNA and endonuclease cleavage on either side of the dimer on the single DNA strand
- helicase, polymerase, and ligase complete the repair
bulky adducts
- aka polycyclic aromatic hydrocarbons
- environment-damaging agents that only interact with DNA when they are activated in our bodies
removal of uracil from DNA
- one of the best understood base excision repair systems
- uracil can arise from incorrect incorporation or deamination of cytosine
- UNG removes dUMP resides in DNA
- enzyme that removes the ribose-phosphate is called deoxyribose-5’-phosphatase
- has to happen so GC doesn’t transition to AT
structure of the uracil-binding pocket of human UNG
- uracil-DNA N-glycosylase
- acts by flipping the uracil base out of the DNA duplex and into a pocket where cleavage occurs
- pocket is small enough to exclude purine bases & prevents thymine from binding between C5 methyl group of thymine and Tyr-147
- doesn’t discriminate between U’s paired with A’s or G’s
BER (base excision repair) of oxidative damage
- 8-oxoguanine is an abundant ROS (reactive oxygen species) oxidation product
- C-oxoG base pair becomes A-oxoG in a second round of replication & A-T after a 3rd round
- both bacteria and humans have BER enzyme system to recognize and repair oxoG bases
- in humans, OGG1 DNA glycosylase recognizes and cleaves oxoG base
- transversion mutation
methyl-directed mismatch repair in E. coli
- mismatch repair enzymes identify the newly replicated strand by its lack of methylation at -GATC- sites by MutS
- MutHLS complex scans the DNA for the nearest 5’-GATC sequence, cleaving to the 5’ of G in the unmethylated strand and excising the DNA back past the mismatch
- DNA pol III and ligase complete the repair
double strand break (DSB) repair through homologous recombination
- genetic defects involving DSB in the BRCA1 and BRCA2 genes are risk factors for breast/ovarian cancer
- DSBs are the most lethal form of DNA damage bc it destroys the physical integrity of the chromosome
- repaired bu homologous recombination or non-homologous (end joining NHEJ)
- NHEJ more efficient if ends can be rejoined precisely at sites of damage
- HR can only occur in S or G2 when a sister chromatid is available
- begin with phosphorylation of a variant histone
daughter strand gap repair
- the replicated portion of an incompletely replicated chromosome is used as a repair template
- process depends on the multifunctional protein, RecA
- damaged site (thymine dimer) isn’t repaired
- allows replication to continue and an excision system comes in later to repair damage
holliday model for homologous recombination
- DNA is nicked at the same site on 2 paired chromosomes
- next partial unwinding of the DNA allows strand invasion
- Enzymatic ligation generates a crossed-strand intermediate (Holliday junction)
- cross-strand structure can move along the duplex
- holiday junction isomerizes
- strand breakage generates a recombinant or non recombinant heteroduplex
RecA
-uses ATP to promote the pairing of homologous DNA sequences
RuvA
- DNA binding protein
- recognizes the Holliday junction
RuvB
- ATP-driven motor protein
- rotates arms of bench in opposite directions to drive branch migration
RuvC
nicks the two strands
gene rearrangements
generates antibody diversity
v-joining
- RAG1 & RAG2 catalyze double strand breaks, initiating recombination
- cutting and splicing may occur anywhere within the terminal trinucleotide, and can generate 4 different recombinant forms
transposon
-bacterial transposable element flanked by short repeated sequences
transposase
catalyzes the reactions involved in transposition
retroviruses
use long terminal repeats (LTRS) for integration into the host chromosome
