New Material Lecture 1/5: Mutation and DNA Repair

what are mutations

changes in nuc sequences that get passed on to daughter cells

• new alleles are created by muts of an existing allele

• muts that occur in germ cells can be passed on to offspring

why are mutations important for life

evolution/adaptation/genetic diversity

why are there mechanisms to fix muts

• lethal muts

• too many muts ruin the integrity of a species

  • no maintenance of key genetic/allele combos

  • eg if trait promotes fitness, parents must be able to mostly pass that onto the offspring

what are substitution muts

replacement of one base by another base

what are purines and pyrimidines

Purines (adenine and guanine) are two-carbon nitrogen ring bases

pyrimidines (cytosine and thymine) are one-carbon nitrogen ring bases

what are transition and transversion muts examples of? explain what each is

The 2 types of substitution muts

transition → purine replaced by purine or pyaimidine by pyrimidine

transversion → purine replaced by pyrimidine or vice versa

what are deletions

one or more bps are lost from dna

what are insertions

one or more bps are added to dna

how many bps are deleted/inserted in those types of muts

can involve one or few to hundreds/thousands of bases

• they’re not ALL frameshifts if multiple of 3

what is a point mut

umbrella term → any mut that is only affecting a single nucleotide (sub, insertion, deletion)

what are frameshift muts

when deletions or insertions change reading frame of gene

• causes AAs incorporated during translation downstream of region where deletion/insertion occurred

• can introduce early stop codons

what are inversion muts? why do they occur

when a segment of dna is reversed (inserted backwards)

• occurs when two dsDNA (double-stranded) breaks occur and excised dna is added back in the incorrect orientation

do all muts lead to diff phenotypes

no → can have silent muts

• a lot of dna does not code for AAs (eg introns)

• coding is redundant so sometimes changes in bps may j result in same AA produced

t/f: spontaneous muts occur at a fast rate

f → they occur at a very slow rate

are muts heritable in euk organisms

only muts in germ cells are

• muts in somatic cells are passed to daughter cells in that individual

are muts heritable in proks

yes → always j make copies of themselves so all muts are heritable

t/f: sperm have more muts than eggs

explain why or why not

true → long-term arrest of meiosis in egg cell precursors during diplotene 1 inc the occurence of non-disjunctiuon during oogenesis compared to spermatogenesis

BUT spermatogenesis is ongoing → occurs en mass so inc chance of muts and passing on those muts to offspring (bc germ cells)

t/f: muts in maternal germ cells greatly inc w age and paternal germ cell muts are relatively constant through life

false → opposite

explain the experiment that showed us if muts occur randomly or bc of environmental stress

• let ecoli cultures grow for specified aount of time

• plated equal portions of each culture w basteriophage T1

  • if muts occured bc of environment, would be around equal proportions of each culture that are resistant (bc muts would ONLY occur after phages were introduced)

  • if random, they varying amounts

• found they were random

• muts occurring early in colony development resulted in many resistant colonies when plated; those occurring later resulted in few resistant colonies

how did replica plating verify that bacterial resistance is result of preexisting muts

agar plate contained colonies of type of bacteria killed by penicillin

• created “stamps” of those cells and transferred it to an agar plate containing penicillin

• most cells would die but cells w resistance would form new colonies

• position of resistant colonies would tell if colonies on OG plate ALREADY had resistance if resistance predated interaction w pen

  • if already had resistance before pen, all colonies on agar plates would be immune in same areas

  • if developed resistance after pen, would be immune in diff areas

• found colonies on agar plates were all resistant in same areas → hence had resistance before pen → hence muts are random

t/f: selective pressures cause mutations

false

what are depurination and deamination? explain them

they are processes that lead to muts

Depurination: hydrolysis (removal) of purine bases

• 1000/hour in every cell

• random bp introduced during repair

Deamination: removal of an amino (-NH2) group

• can change C to U

• after replication, normal C-G pair becomes an A-t pair

  • bc there is no U in dna, it becomes a T when replicated and A is then paired w it

which types of muts do depurination and deamination result in

• substitution muts

what are natural muts

mistakes in dna sequence that occur at a low freq all the time

• natural processes

what are induced muts

any physical or chem agent that raises the freq of muts above the spontaneous rate

• (eg x-rays, some viruses, etc)

what can excess active oxygen species in cells lead to

oxygenation of nucleotides → O2 binds to guanine, makes it able to pair with adenosine, turns that bp from G-C to A-T

• substitution mutation

what do x-rays to do dna

break the dna backbone (results in deletion, translocation (swapping positions w another broken segment), or inversion)

what does UV light do to dna segments

produces thymine dimers (creates bond between 2 thymines, changes double helix structure, dna pol cant work on that region anymore :. transcription j stops, dna cant be duplicated)

t/f: all mistakes in DNA become integrated into new cells after replication

false → dna repair mechanisms fix most mistakes before they can be replicated

what is the first step in resolving mistakes in DNA? explain it

proofreading

• proofreading portion of DNA pol is a 3’ to 5’ exonuclease

  • fixes most mistakes made by pol → reducing error rate to be even less than usual

what does base excision repair do? how does it do that

removes lesions of damaged DNA

• DNA glycosylases removes altered nitrogenous base and therefore nucleotides

• new DNA synthesizes to fill in the gap

what does nucleotide excision repair do

corrects damaged nucleotides

• UvrA and UvrB complexes → scan for distortions in the double helix (eg those produced by thymine dimers)

• UvrB and UvrC complex → cut around the damaged DNA

• dna pol fills in the gaps

what can unrepaired dsDNA breaks lead to

deletions and chrom rearrangements

what are homologous recombination and non-homologous end-joining (NHEJ)

the two ways dsDNA breaks are repaired

• active during all parts of the cell cycle (not just dna rep)

• two repair mechanisms

  • homologous recombination: similar process to crossing over during meiosis

  • non-homologous end-jining (NHEJ): repair of double-strand breaks without using a template

t/f: larger breaks in dna inc chances there is still a problem after the dna is repaired

true

what is the SOS system in bacteria and microhomology-mediated end-joining (MMEJ)

the last resorts to fix mistakes during replications → the error prone repair syetsms

• they will introduce errors but not MASSIVE ones

SOS system in bacteria

• used at replication forks that stalled bc of unrepaired dna damage

• “sloppy” dna pol used instead of normal pol

• adds random nucs opposite to damaged bases

Microhomology-mediated end-joining (MMEJ)

• similar to NHEJ but nucs are removed at dsDNA breaks to prod short complementary regions, leading to small deletions

if mistakes aren’t caught during dna replication, how do repair enzymes know which strand is wrong

Methyl-directed mismatch repair

what is Methyl-directed mismatch repair

• bacterial recognition and repair system that can fix muts AFTER dna rep

  • relies on “tagging” parental strands w methyl groups

• euk cells also have a mismatch repair system but the “tag” is not known yet

what happens is there is too much damage to a cell at once

apoptosis → programmed cell death

• too much damage to repair

  • eg epithelial cells dying after too much UV exposure (sun burn, peeling)

• some muts derail apop so some damaged cells don’t die (common in cancer cells)

what is xeroderma pigmentosum

• mut in any one of the seven genes involved in nuc excision repair

what are hereditary forms of colorectal cancer

muts in mismatch repair genes

what are hereditary forms of breast cancer

muts in BCRA1 and BCRA2 involved in dsDNA break repair by homologous recombination