Ch 12: DNA mutation and repair

normal biological/chemical processes

spontaneous mutations happen randomly and are usually linked to

eukaryotes (then prokaryotes then phages)

spontaneous mutation rate is highest in ___

replication errors, DNA damage, chromosomal rearrangements

3 main causes of mutations

transition

pyrimidine replaces pyrimidine or purine replaces purine

transversions

purine replaces pyrimidine or pyrimidine replaces purine

frameshift mutations

adding/deleting nucleotides shifts the reading frame

truncated protein

shift in reading frame uncovers a stop codon

initial reading frame (mutation not as severe)

adding/deleting multiple of 3 nucleotides reestablishes __

template slippage

• DNA pol temporarily dissociates from DNA when there are short repeated sequences resulting in adding/deleting some repeats

• diagnosed using PCR

Huntington’s disease

• autosomal dominant disease causing progressive neurodegeneration

• caused by trinucleotide expansion from template slippage (CAG - Gln)

• symptoms appear midlife

fragile X syndrome

• most common cause of inherited mental retardation (1/1500 males)

• caused by trinucleotide expansion from template slippage (CGG - Arg)

Huntington’s disease

disease caused by trinucleotide expansion from template slippage of CAG repeat (Gln)

fragile X syndrome

disease caused by trinucleotide expansion from template slippage of CGG repeat (Arg)

FMR1

• CGG repeat of fragile X syndrome is just upstream of this gene

• encodes for protein involved in regulation of translation

normal

6-54 copies of fragile X syndrome CGG repeat

transmitting males (full mutation to grandchildren)

52-230 copies of fragile X syndrome CGG repeat

affected (inactivates FMR1)

230-2300 copies of fragile X syndrome CGG repeat

meiosis in females

fragile x syndrome repeat expansion only happens during ____

inversion

single chromosome mutation where a segment of the chromosome inverts

not viable

meiotic products that crossed over within a heterozygous inversion are ___

heterozygous inversion

chromosome inversion in only one of the two homologous chromosomes

viable

meiotic products with no crossovers within inverted region of chromosome are ___

effect of crossover within an inversion (if those genes within inversion are separated → nonviable)

• keeps a set of genes for an adaptive (helps it survive) trait together

• genes for multiple queens and aggressive behavior in fire ants are contained within an inversion relative to other ant species

reciprocal translocations (can’t be regulated when translocated to other chromosome)

• segments of genetic material are swapped between two NONhomologous chromosomes (no genetic material is gained or lost)

• ex) Philadelphia chromosome

Philadelphia chromosome

• normal ABL proto-oncogene on chrom 9 is tightly regulated

• BCR-ABL oncogenic fusion protein of chrome 22 (philadel) is not regulated

proto oncogene

normal gene that regulates cell growth/proliferation but has potential to contribute to cancer development if expression is altered

chronic myelogenous leukemia (CML)

95% of individuals with this disease have Philadelphia chromosome reciprocal translocation

Gleevec (imatinib)

• drug designed to inactivate BCR-ABL fusion gene in Philadelphia chromosome resulting from reciprocal translocation (chronic myelogenous leukemia)

• kills cancer cells but doesn’t harm normal cells bc normal cells don’t have chrom 9/22 translocation

• resistant cells to this drug targeted by second generation drugs

ethyl methanesulfonate (EMS)

mutagen that chemically modifies DNA by causing transversions

intercalating agents

mutagens that cause frameshift mutations, inhibit transcription, and inhibit DNA replication

UV light (50-100 per cell per second in sunlight)

mutagen that cause pyrimidine dimers

ionizing radiation (X-rays, gamma rays)

high energy mutagen that causes double strand breaks

10⁶ or 10⁸

after base selection and proofreading, mistakes occur every ____ bp

base analogs (uracil has more tautomeric shifts)

mutagens with tautomeric shifts (more tautomeric shifts=higher mutation rate)

reactive oxygen species (hydrogen peroxide, hydroxide, superoxide radicals modify bases)

• byproducts of normal cellular metabolism or exposure to high energy radiation

• causes oxidative damage

8-oxoguanine

• most common oxidative damage product

• can base pair with A or C

• associated with many human cancers (lung cancer)

depurination and deamination

• common cause of DNA damage

• 10,000 purines lost per day in typical human cell

• 100-500 cytosoines deaminate per day in typical human cell

hydrolytic damage

water damages by deaminating cytosine → uracil (which base pairs with A)

DNA

• only molecule that is repaired by the cell

• bad RNA/protein → just make a new one

1,000

how many DNA lesions per day in E.coli

100,000

how many DNA lesions per day in eukaryotes

it will become permanent (a mutation)

what happens if replication errors aren’t fixed quickly (before replication)

Dam methylase

recognizes GATC and methylates the A

MutS (S for scan)

recognizes alteration in DNA backbone due to bump created by mismatch

MutL

once MutS finds a mismatch, it forms a complex with ____

MutS-MutL (L for loop)

this complex scans DNA for Dam site bidirectionally, forming a loop

MutH

• once MutS-MutL complex finds Dam methylase GATC site, it recruits ____(nickase) to cleave new strand

• then helicase unwinds and Pol I exonuclease degrades past the mismatch

DNA pol III

fills the gap in mismatch repair

MSH2/MSH6

MutS homologs in eukaryotes

MLH1/PMS2

MutL-like proteins in eukaryotes (no hemi-methylated DNA)

PCNA (sliding clamp)

MSHs (MutS homologs in eukaryotes) interact with ____ and it guides MSH to growing DNA strand for repair

inherited cancer susceptibility

mutations in mismatch repair enzymes leads to

DNA photolyase

• uses visible light to directly reverse UV-induced pyrimidine dimers

• not present in mammals

• energetically cheap

photon (from visible light)

chromophore in photolyase absorbs ___ and passes excitation energy to FADH^-

FADH^-

excited photolyase chromophore passes energy to ___

pyrimidine dimer

FADH^- (from DNA photolyase) donates electron to rearrange bonds in _____

methyltransferase

• directly reverses O⁶-methylguanine back to guanine (binds to thymine instead of cytosine)

• energetically expensive because enzyme is modified irreversibly (can only do reaction once)

specific step of base excision repair

DNA scanned by specific glycosylases and different types of damaged bases are removed

specific glycosylases (uracil DNA glycosylase deaminates C→U et al.)

• scans DNA in base excision repair and removes damaged bases

• leaves abasic site (empty)

10,000

how many purines are spontaneously lost per day per cell?

apurinic (AP) sites

10,000 purines spontaneously lost per day per cell creates abasic sites AKA ____

glycosylases

flips each base out one by one and binds it to its active site to check for damage in base excision repair

general step of base excision repair

abasic sites recognized → backbone cleaved → short segment resynthesized

AP endonuclease

recognizes abasic sites in base excision repair and cleaves backbone

Pol 1

resynthesizes short segment in base excision repair in BACTERIA

Pol 1

extends from 3’ end of abasic site and displaces strand because no forward exonuclease in base excision repair in EUKARYOTES

flap endonuclease

cleaves displaced strand (flap) in base excision repair in EUKARYOTES after Pol 1 extends from 3’ end

Pol beta

adds one nucleotide to abasic during base excision repair in EUKARYOTES

nucleotide excision repair

removes bulky lesions like pyrimidine dimers (major repair pathways for humans since we don’t have photolyase)

UvrAB (Uvr = UV resistant)

scans dsDNA for distortion caused by damage → melts short DNA region → UvrA is released leaving only UvrB in nucleotide excision repair

UvrC (excinuclease)

nicks both sides of distortion where UvrB is bound in nucleotide excision repair

UvrD

helicase that removes the fragment cleaved by UvrC in nucleotide excision repair

Xeroderma Pigmentosa

• disease where cells are extremely sensitive to UV light

• causes pyrimidine dimers and oxidative damage

• leads to various cancers like melanoma

• 40% die before age 40

transcription coupled repair

• stall RNA polymerase to facilitate nucleotide excision repair

• damage such as thymine dimer stalls RNA polymerase passage

• stalled RNA pol recruits nucleotide excision repair proteins

• ensures that transcribed strands are repaired quickly

Cisplatin

• drug that forms cross links with N7 of two purines

• blocks replication fork and RNA pol → apoptosis

• useful on cancer cells

translesion (bypass) synthesis

• if DNA pol III is stalled due to lesion, one of translesion pol replaces it on beta-sliding clamp

• error prone but adding anything is better than doing nothing

• pol III takes over after lesion is passed

at least 9

how many specialized DNA polymerases in humans?

mutagenic translesion synthesis

E. coli pol IV and V and most translesion enzymes in eukaryotes insert random nucleotides (anything is better than nothing)

non-mutagenic translesion synthesis

• when DNA pol is stalled, yeast Rad30 and human XPV only bypass thymine dimers → only insert AA in complementary strand

• error-free translesion synthesis