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MUTATION JARGON GENE MUTATION = POINT MUTATION

(scale of mutation is small and is localized to a specific region, a single nucleotide or a few adjacent base pairs)

↓

at the DNA level: Ë single base pair substitutions: transitions & transversions Ë single (or a few) base pair addition or deletion: indels

Ë gene mutation by transposon insertion at the level of at the protein gene expression: level: promoter mutations nonsense splicing mutations missense regulatory mutations [neutral] silent frameshift at the level of gene function: loss-of-function gain-of-function [neutral]

CHROMOSOME MUTATION • involves segments of chromosomes or

whole chromosomes or whole genomes • alterations in chromosome structure

and number • deletion, duplications, translocations

and inversions • CNVs: copy number variations

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Mutation: any heritable change in the genetic material (excludes changes caused by normal recombination events)

S--t happens The integrity of genomic DNA is constantly under threat, even in perfectly healthy cells. DNA damage can result from the action of endogenous reactive oxygen species, or from stochastic errors in replication or recombination, as well as from environmental and therapeutic genotoxins.

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Spontaneous mutations: a mutation that occurs in the absence of known mutagens • uncorrected errors* that occur during DNA replication, repair

or recombination • spontaneous lesions that occur to the DNA molecule under

normal physiological conditions and that are not repaired by the cell’s DNA excision repair processes -- see text for examples of excision repair process

* what quality control processes are in place?

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Here’s what happens if an error slips by the quality control processes:

After two rounds of replication, the mutation is present in both strands

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Transition mutation: a specific type of mutation that involves the replacement of a purine with a purine or a pyrimidine with a pyrimidine:

for example a GCèAT change

______ G____ _____ A____ ______ C ____ è_____ T____

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Spontaneous mutations: a mutation that occurs in the absence of known mutagens • uncorrected errors that occur during DNA replication, repair or

recombination • spontaneous lesions that occur to the DNA molecule under

normal physiological conditions and that are not repaired by the cell’s DNA excision repair processes -- see text for examples of excision repair process

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Your DNA is under constant assault: Science Dec. 23, 1994

• Every second that you read this, the DNA in each cell of your

body is being damaged • Chemical bonds are breaking • DNA strands are snapping • Nucleotide bases are flying off • Each cell loses more than 10,000 bases per day just from

spontaneous breakdown of DNA at body temperature • Meanwhile many cells are dividing and therefore copying DNA

and each copy introduces the possibility of error • Exposure to carcinogens adds to the injury and causes strange

new forms to sprout from the double helix

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(After T. Lindahl, Nature 362:709–715, 1993)

The sites on each nucleotide that are known to be modified by • spontaneous oxidative damage (red arrows) • hydrolytic attack (blue arrows) • uncontrolled (non-enzymatic) methylation by the methyl group donor S-

adenosylmethionine (green arrows) • WIDTH of each arrow indicates the relative frequency of each event

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Rate of depurination: 4 X 10-9 sec-1 at pH7.4 It may then be predicted that an E. coli cell, growing with a generation time of 40 min at 37degC, should lose 0.5 purine/chromosome in each generation. For a mammalian cell, which contains ~800 times more DNA than E. coli and grows with a generation time of 20 hr, 12,000 purines should be lost from the DNA in each cell generation due to hydrolysis. However, as at least 50% of the DNA is present as nucleohistone in this case and therefore may be protected, it is possible that 2,000-10,000 depurination events per generation would be a better estimate. In the same fashion, a long-lived, non-growing mammalian cell, e.g., a human nerve cell would lose ~108 purines from its DNA during the lifetime of the individual, or ~3% of its total amount of purine residues in DNA: ARGGHH: REPAIR NEEDED BIOCHEMISTRY, VOL. 11, NO. 19, 1972

Depurination is a very common spontaneous lesion It is estimated that a single mammalian cell loses 10,000 purines from its DNA in a 20 hour period (see below)

Spontaneous loss of guanine results in an abasic site

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Most AP sites are repaired by the base excision repair system, which deals with small scale lesions that don’t distort the helix Uracil DNA glycosylase flips a uracil residue out of the duplex, shown in yellow.

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Unrepaired depurination of DNA results in abasic sites -- a common spontaneous lesion spontaneous loss of G from ribose AP = abasic = apurinic/apyrimidinic

LESION REPAIRED SEE figure 15-23 in 9th or 16-20 in 10th: Base excision repair

---------G----------- ---------C-----------

--------- (AP)---------- -----------C-----------

LESION NOT REPAIRED and cell entere S PHASE

3’ ----------(AP)--------------5’ 5’----------| <----G---------------- 5’ 5’--------------C-----------------3’

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So-called bypass polymerases can move past a lesion in the template High fidelity replicases cannot

spontaneous loss of G from ribose ------G -------- ---------(AP)--------- ------C--------- ---------C--------- repaired* --------(AP)--------- --------- A--------- **bypass polymerases will insert a purine opposite an abasic site ---------G -------- -----------T---------- ---------C--------- --------- A---------

DNA replication occurs: unrepaired strand is template for one of the daughter strands another round of

replication

*SEE figure 15-23 in 9th or 16-20 in 10th: Base excision repair ** SEE figure 15-27 in 9th or 16-24 in 10th

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Transversion mutation a specific type of mutation that involves the replacement of a purine with a pyrimidine or a pyrimidine with a purine: for example a GCèTA change

______ G____ _____ T____ ______ C ____ è _____A____

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Addition or deletion of one or a few bases can occur spontaneously: such a lesion typically results from “strand slippage” during DNA replication

Recall triplet repeat expansion and contraction in Fragile X

Also, file this info away and retrieve during our discussion of DNA fingerprinting

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The DNA sequences shown above are more prone to strand slippage than, say, the sequence shown below: 3’ CTAGACTCAGTGTA….5’ template strand

5’ GATCTGA…..3’ daughter strand Why?

Intercalating agents (see pg 5) induce mutations that involve the addition or deletion of a small number of base pairs: see pg 528 of 9th and 565-56 of 10th

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mutagen: any agent that increases the mutation rate above the background, or spontaneous, rate induced mutation: mutation occurring in the presence of a mutagen base analog mutagen: chemically resemble one of the purine or pyrimidine bases Because of their close chemical similarity to the bases naturally found in DNA, base analogs can be incorporated into DNA during replication

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5-bromo-uracil is a thymine analog

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This mutagen can also cause GC à AT transitions Draw out how this would occur

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But the naturally-occurring bases have different forms and can mis-base pair Why then do base analog agents act as mutagens?

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S k u l l w i t h B u r n i n g C i g a r e t t e

A r t i s t ?

• Benzo[a]pyrene is a compound found in cigarette smoke

• Hydrocarbons like benzopyrene are insoluble in water

• In the liver and lungs such compounds are chemically modified to make them more soluble so the body can excrete them.

• Intermediates that are generated include epoxides that react with guanine

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The soluble product of the reactions includes benzo(a)pyrene 7,8 diol-9,10 epoxide, which can attack the electron rich area of a DNA base The enzyme oxygenase catalyzes steps 1 and 3 The lungs have a system for inducing synthesis of oxygenase when levels of benzo(a)pyrene are high

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Cigarette smoke carcinogens such as benzo[a]pyrene are implicated in the development of lung cancer: • about 60% of human lung cancers involve mutations in a tumor

suppressor gene called p53 • a mutation database exists that includes more than 500 entries of

sequenced p53 genes from lung cancer cells • a large percentage of these 500 entries have GC to TA transversions

(see next page for mechanism) • such mutations are hallmarks of mutagenesis involving polycyclic

aromatic hydrocarbons

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What happens to the modified G*? 1. The defective base is hydrolyzed (either spontaneously or via a repair

process; this leaves a deoxyribose with no base attached -- an apurinic site 2. If the apurinic site is not repaired before the next round of DNA

replication, an A typically is inserted opposite the “empty site” 3. After another round of DNA replication, the GC base pair has been

converted to a TA base pair 4. See diagram next page

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Unrepaired abasic sites * = benzo(a)pyrene 7,8 diol-9,10 epoxide

------G *-------- #1 ---------(AP)--------- ------C--------- ---------C--------- repaired (AP)=abasic site #2 ---------(AP)--------- --------- A--------- bypass polymerases will insert a purine opposite an abasic site #3 ---------G -------- ----------T---------- ---------C--------- --------- A---------

#2 DNA replication occurs: unrepaired DNA strand is template for one of the daughter strands #3 another round

of replication

a pack a day

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