mutation: basic features of the processbio.classes.ucsc.edu/bio105/winter12/notes/12 13...

Bio 105 Mutation 2/12/12

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MUTATION: BASIC FEATURES OF THE PROCESS GENERALIZATION Mutation of a DNA target sequence typically is a sequential process with defined intermedaites (premutagenic lesions). Elaborate, specific mechanisms have evolved to repair lesions. Most lesions are "fixed" , ie. become mutations, only after replication. Some mutations are actually introduced by an error-prone DNA repair process. Thus, replication collaborates in the mutation process.

DNA Protein Phenotype Base Pair Substitution

Silent Missense Nonsense

Neutral Alteration of Function Null

Indel Frameshift Mutation: Spontaneous or Induced SPONTANEOUS vs INDUCED (Intrinsic vs Extrinsic) MUTATIONS The distinction between "spontaneous" and "induced" mutations is based on an operational definition of induced mutations as those which result from intentional exposure of an organism by an investigator to chemicals, radiation or other mutagenic treatment. Spontaneous mutations, then, are those which occur "naturally" in the environment without intervention of an investigator. By these definitions spontaneous mutations could result from the same type of molecular processes as induced mutations because there are naturally occurring environmental mutagens and naturally occurring sources of radiation. A more meaningful distinction can be drawn between those mutations which result from the interaction of DNA with some external influence (such as chemicals or radiation) whether or not they were intentionally applied by an investigator and those which result from inherent properties of DNA itself or from essential attributes of DNA replication. These latter would be "spontaneous" in a non-trivial sense, although admittedly some mutagenic treatments act by amplifying the rate of

TARGETSEQUENCE

PREMUTAGENIC LESION/DAMAGED DNA

"spontaneous" or induced repair

LETHAL

fixation of sequence alteration (DNA replication)

SOS error-prone repair

ALTERED SEQUENCE

PHYSIOLOGICAL EXPRESSION OF MUTANT PHENOTYPE

LETHAL?


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spontaneous events or by interfering with mechanisms that repair spontaneouly occuring premutagenic lesions. The most important types of spontaneous processes leading to mutation are: •misincorporation of bases during DNA replication (tautomeric shifts) •depurination •deamination of cytosine or (less commonly) adenine •small additions and deletions (indel mutations) due to "slipped mispairing" •insertional inactivation by transposable elements THE MOLECULAR BASIS OF MUTATION BP Substitutions Base Pair Substitutions are one class of what the text calls “Point Mutations”. The other class of point mutation is the “indel” class. The term indel refers to changes by addition or deletion of one or several base pairs. Base pair substitution implies a change in the identity of a single base pair with no change in the total number of base pairs. There are 2 flavors of bp substitution

Transition replacement of one purine by the other purine (this entails replacement of one pyrimidine by the other) Transversion replacement of a purine by a pyrimidine (this entails replacement of a pyrimidine by a purine) For each of the 4 base pairs 2 transversions and one transition are possible. Example: AT ---> GC = transition

AT ---> CG = transversion AT ---> TA = transversion

(Note that AT ---> GC, TA ---> CG, A ---> G, and T ---> C all designate the same type of bp substitution.) The effect of a base pair substitution on the phenotype may range from neutral to lethal depending on the genomic and environmental contexts. Let’s consider the effect of bp substitution within a coding sequence (gene), on the amino acid sequence of the protein product of the gene. BP substitution in a coding sequence may be either silent, missense or nonsense.

● Silent substitutions change an amino acid codon to a synonomous codon. The sequence of the protein is unaltered.

● Missense substitutions alter a codon for one amino acid to a codon for a different amino acid.

This changes the identity of one amino acid in the protein sequence. ● Nonsense substitutions alter an amino acid codon to one of the three termination codons. The

protein product in this case is only a shortened amino terminal fragment of the original and that usually has no function.


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● BP substitutions that convert a termination codon to an amino acid codon have no specific

designation that I am aware of. Excision Repair SINGLE STRAND GAP REPAIR in E. coli Several types of premutagenic lesion are each targeted by specific excision repair mechanisms that remove a segment of a DNA single strand containing the lesion. The ss gaps created by various excision mechanisms are repaired by a common ss gap repair mechanism involving the sequential action of DNA Polymerase I and DNA ligase.

PRE-MUTAGENIC LESION

TARGET DNA

5' 3'

5' 3'3' OH 5' P

5' 3'3' OH 5' P

Removal of lesion in onestrand; various excisionrepair mechanisms.

DNA Polymerase I

DNA Ligase

SS GAP

SS NICK

REPAIRED


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Base Pair Substitutions by DNA Base Tautomerism Instability of the DNA base ring structures is the underlying molecular basis for most nucleotide misincorporation during replication. All 4 bases are subject to a kind of "molecular identity crisis" referred to as tautomeric shifting. The phenomenon of tautomerization is usually presented in introductory Organic Chemistry classes; where the profound significance for molecular genetics and evolution is often overlooked. Tautomerization means that a molecular structure rapidly equilibrates between 2 different forms with the same molecular formula, but with a different covalent structure. ( Linguistically, tautomer refers to a needless or redundant restatement of fact in different words.) Aromatic ring structures like the DNA bases are prone to tautomerize. The diagram shows the equilibrium between the two tautomeric forms of thymine, and compares them to the ordinary form of cytosine. (See also Fig. 13.9 and Fig. 13.10)

Note several important aspects: 1. The thymine tautomer on the left is called “keto” after the C4=O in the ring, the tautomer on the right is

called “enol” after the C4-OH. 2. The equilibrium lies to the left. So, the keto form is the “ordinary” form shown in all the textbooks. Any

given thymine molecule exists as the keto form most of the time; excursions into the enol form are infrequent and fleeting. But in any population of thymines, a small percent will inevitably be in the enol form at any given time.

3. The arrows in the diagrams indicate ability of atoms to hydrogen bond. Arrows point away from H

donors and towards H acceptors. 5. The keto and enol forms of thymine have different H-bonding properties, as shown by the arrows. In

fact, the enol form of thymine mimics the H-bonding properties of C and will form a complementary base pair with G rather than with A! So the statement “T pairs with A’ must be modified to “T pairs with A “ as long as both bases are in their "ordinary" tautomeric forms".

Remarkably, each of the four DNA bases can exist in either of two tautomeric forms. Even more re,arkable, the tautomeric shift causes the base to acquire the base pairing properties of its cognate base (i.e. the rare tautomer of G pairs with the predominant tautomer of T but not with the predominant tautomer of C, etc.).


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In Fig. 13.10b they present a specific scenario in which a G* in the template strand pairs with a dTTP during replication. After 2 subsequent rounds of DNA replication, the base pair mismatch (G with T) is fixed as a GC --> AT transition in 1/4 progeny helices. The diagram is misleading where it suggests that G would pair with T after it shifts back to the keto form. It should be something like:

The distortion of the helix caused by the GT mismatch is essential for the repair process described below. Be certain that you can deduce the character of the BP substitution that would result from tautomeric shifting of any DNA base either as a template base or as in dNTP precursor. i.e. Fill in 7 blank boxes in the table below following the example.

Base Shift as Template Base

Shift as Precursor Base

A

T

G

GC → AT Fig. 13.10

or Fig. 13.2

C


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The equilibrium constants for the base tautomerizations are used to estimate that the misincorporation rate should be as high as 10-4 errors per base per replication (i.e. 1/10,000). This would amount to 400 BP substitutions in the E. coli genome at every replication. The actual error rate observed in wild-type E. coli is only 10-8 because 99.99% of all mismatches introduced by tautomerization are subsequently repaired by the 2 mechanisms described below. PROOFREADING Proofreading based on 3' --> 5' exonuclease activity of many DNA polymerases (See Sec. 10.2.3 Proofreading Activities of DNA Polymerases). (METHYL-DIRECTED) MISMATCH REPAIR This is described in Sec. 13.6.3: Other DNA Repair Mechanisms, but no diagram is offered to accompany the text. Following replication, methyl-directed mismatch repair can excise an incorrect base specifically from the newly synthesized strand of DNA.

Dam DNA methyltransferase; methylates N6 position of Adenine in all GATC sites; dam-

has mutator phenotype specific for transitions. MutS binds to DNA containing Normal Base Pair Mismatches (heteroduplex) except C-C;

mutS- has mutator phenotype. MutL binds to MutS/heteroduplex complex; mutL- has mutator phenotype. MutH latent endonuclease incises unmethylated strand of a hemimethylated GATC

sequence; activation requires proximal MutS/MutL/heteroduplex complex and ATP hydrolysis; mutH- has mutator phenotype.

Mut U helicase Exo I (5' --> 3') OR Exo VII (3' --> 5') ss excision following MutH incision

Several kb of the unmethylated strand is excised, followed by ss gap repair. Similarities to Type II restriction/modification systems!


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Spontaneous Cytosine Deamination Cytosine is the least stable of the four bases in nucleic acids and undergoes "spontaneous" oxidative deamination at a rate of 5 x 10-13 sec-1 in double-stranded DNA (1, 2). At this rate, 40 to 100 deamination events should occur in a human genome per day. Deamination of cytosine produces U, leading to GU abnormal base pair mismatches. The GU mismatch will be fixed as a GC to AT transition substitution following two subsequent rounds of DNA replication. See Fig. 13.155 or 13.86 A repair enzyme, Uracil DNA glycosylase, can excise U from the GU mispair by cleaving the glycoside bond. The resulting AP site is then a substrate for the AP endonuclease excision and ss gap reapair pathways (discussed in the text) which restore the original target GC. The text also briefly notes the possibility that T occurs in DNA, rather than U, because it creates a context for efficient repair of AU lesions. (See discussion of 5-meC below.) C:G to T:A transitions dominate the spectra of spontaneous base substitutions in Escherichia coli and in mammalian cells (5, 6, 7, 8). In primates, C:G to T:A transitions are thought to account for over 40% of all base substitutions within the globin cluster (9, 10). These data, and the high rate of GC to AT transitions observed at sites of 5-meC residues (below) argue for the significance of deamination in mutagenesis.

GC

U

G

U

AU

G

G

AT

Specific BASE Excision by!Uracil-DNA Glycosylase

"AP" Site

AP NUcleotide ExcisionGap Repair!DNA Polymerase and Ligase

Replication ReplicationDeamination

Hydrolytic deamination of cytosine is thought to be initiated by the addition of a water molecule across the 5, 6 double bond followed by electronic rearrangements that create an iminium group that is susceptible to hydrolysis (3, 4). In double-stranded DNA, access to position 6 of cytosine is restricted by the deoxyribose sugar, and this restricts the ability of water to attack C6 of cytosine to initiate deamination. Cytosines in single-stranded DNA deaminate to uracils at 140 times the rate for cytosines in double-stranded DNA. Thus, cellular processes, such as transcription and replication that render DNA single-stranded should promote C to T mutations. Beletskii and Bhagwat ( 11 ) have documented a four-fold increase in the frequency of C to U transitions in the non-transcribed strand of the E. coli tac promoter.


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There is considerable interest in the idea that genes that are highly transcribed may accumulate more spontaneous mutations (12, 13, 14). The results presented here support this idea and predict that during evolution, genes should acquire more C to T mutations in the nontranscribed strand. Recently, this was shown to be correct for some enterobacterial genes (15). When the sequences of several genes were compared among numerous E. coli and Salmonella enterica strains, more C to T than G to A mutations were found in the coding strand. Thus, it may be that the genes whose products are in greatest demand in the cell are most susceptible to spontaneous mutations following deamination. 5-meC Deamination The rate of deamination of 5-methylcytosine (5meC) in DNA is 2 to 4 times higher than that for cytosine. Additionally, deamination of 5meC creates GT normal base pair mismatches that are more difficult to repair than GU mismatches. (Though at least one example of a GT* specific glycosylase has been reported.) Specific C residues are methylated to 5-meC in the DNA of most, if not all, organisms. (Many bacterial restriction/modification methylases, for example, create 5-meC residues.) Several studies have shown these 5-meC residues to be hotspots for base substitution mutations in organisms as diverse as E. coli and humans. In human DNA, approximately 1% of the bases are 5-meC, and the vast majority of these occur in 5' CpG sequences. An estimated 30% of germ-line point mutations associated with inherited human genetic disorders are GC to AT transitions at CpG sequences. Furthermore, there is strong circumstantial evidence that deamination of 5-meC at CpG sequences is responsible for a large fraction of somatic point mutations in the p53 tumor suppressor gene (16). It may be appropriate to consider 5-meC as an endogenous mutagen and carcinogen. Nitrous Acid Nitrous acid elevates the level of cytosine deamination, and also produces a significant level of adenine deamination. The deamination product of adenine is hypoxanthine, which pairs with cytosine (See Table 19-19). Thus, nitrous acid is a bidirectional transition mutagen. UV-Induced DNA Damage and Repair Bacteriologists discovered in the 19th Century that direct sunlight exposure was lethal to bacteria and other microorganisms. Subsequent studies showed the lethal action of sunlight to be primarily attributable to the UV portion of the spectrum between 250-260 nm. This corresponds to the Amax for the DNA bases (whereas the Amax for proteins is near 280 nm). UV irradiation produced by (germicidal lamps) is has been used a method for disinfection since the early 20th Century. UV absorbance (A260) is a common and simple assay for [DNA] in relatively pure samples (50 ug/ml = A = 1.0). A260 increases 40% in ss relative to ds DNA (hyperchromic shift).


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UV mutagenicity (as opposed to lethality) for bacteria was demonstrated in 1914 by V. Henri, 13 years before Muller’s demonstration of X-ray mutagenesis in Drosophila. Henri's discovery was not followed up until the advent of bacteriophage genetics in the 1940’s. Then, Demerec confirmed UV mutagenesis in E. coli by demonstrating a 103X enrichment of bacteriophage T1-resistant mutants in a population exposed to UV. DNA Damage Caused by Short Wave UV UV-induced DNA lesions primarily involve covalent crosslinking of two adjacent pyrimidines in the same strand. The best known, and most common photoproducts are cyclopyrimidine dimers (CPD’s) involving adjacent thymines (i.e. the so-called "thymine dimer"). Another photoproduct is a 6,4 linkage of adjacent pyrimidines.

These crosslinked bases are the principal reason that exposure to short wave UV is lethal to cells. DNA replication complexes stall at these damaged bases, blocking cellular reproduction. Along with other types of DNA damage caused my certain chemicals, these altered bases are often referred to as "non-coding lesions". Molecular biologists sometimes need to be aware that UV-induced DNA crosslinking occurs to some extent when they visualize ethidium-complexed DNA in agarose gels on a UV transilluminator. It is fascinating to contemplate the predicament of organisims on the early earth, who were potentially exposed to intense solar UV radiation flux. The atmospheric ozone layer responsible for blocking short wave UV did not exist for the first several billion years of earth history. We should not be surprised to find that organisms have evolved multiple, redundant molecular strategies to


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repair, or to overcome UV damage. I believe it is correct to claim these survival strategies were all discovered and first characterized by studying bacteria, particularly E. coli. Direct Repair of UV-Induced DNA Damage by Photolyases See Sec. 13.6.1 Light-Dependent Repair “Photoreactivation” is the term suggested by Delbrück for a phenomenon reported by Kelner (1949) in Streptomyces griseus. Following a UV exposure sufficient to reduce survivors to 10-5, a subsequent, immediate exposure to visible light raises survivors to 10-1. Photoreactivation requires the visible light exposure AFTER the UV exposure. The productivity of visible exposure decreases to background over 2 hours in E. coli. Photoreactivation is due the activity of a class of enzymes known as CPD photolyases. See Fig. 13.22 of the text. Properties of CPD Photolyases:

• Single subunit protein. • Bind to CPD lesions in dark. • Catalyze reversal to native structure following exposure to 370 nm light. • 2 Prosthetic groups: FADH•- (semiquinone form); EITHER 5,10-methylenetetrahydrofolate (MTHF) OR 8-hydroxy-5-deazaflavin as the "antenna chromophore".

Nucleotide Excision Repair (see Fig. 13.24 of the text) Many versions of DNA nucleotide excision repair (NER) mechanisms are known, with specificity for various types of lesion. Excision repair of TT dimers in E. coli was the first such mechanism characterized.


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The UvrABC "exinuclease" complex binds to DNA specifically in the vicinity of pyrimidine dimers and makes 2 single strand cuts in the dimer-containing strand on either side of the lesion. With the aid of UvrD (helicase II), a short fragment containing the dimer is released. The resulting gap in the DNA helix is repaired by a generic scenario involving DNA Polymerase I and DNA ligase. NER of pyrimidine dimers in E. coli is accomplished by the sequential activity of 7 proteins. First, a UvrA-UvrB complex scans the DNA, with the UvrA subunit recognizing distortions in the helix caused by pyrimidine dimers. When the complex recognizes such a distortion, the UvrA subunit leaves and is replaced by a UvrC protein. UvrB cleaves a phosphodiester bond in the dimer-containing strand 4 nucleotides on the 3' side of the DNA damage, and UvrC cleaves a phosphodiester bond 8 nucleotides on the 5' side. Next, DNA helicase II (sometimes called UvrD) removes the dimer-containing fragment by actively breaking the hydrogen bonds between the complementary bases. The resulting 12-nucleotide gap in the DNA helix is repaired by a generic scenario involving DNA Polymerase I and DNA ligase. Xeroderma pigmentosa (=Xerodoma pigmentosum) refers to a group of closely related human clinical conditions related to hereditary insufficiency of CPD excision repair. The incidence of XP is 1/250,00, and is inherited as an autosomal recessive. In severe manifestations, an individual's activity must be severely limited to avoid sunlight exposure, and life expectancy is reduced by an extraordinarily high rate of skin cancers. 7-9 different human XP genes have been identified, most of which contribute to the excision of CPD dimers. Introduction of a recombinant CPD photolyase gene (from bacteria?) to XP individuals has been suggested as possible candidate for human gene therapy.


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The "SOS Response" See p. 368. Non-coding lesions that are not repaired directly, or removed by NER, may block progress of a normal DNA replication fork complex, leading to cell death. The SOS response is a strategy to avoid this fate - "Plan C" so to speak. In the SOS response, replication forks are allowed to progress on templates containing non-coding lesions ("translesional synthesis"). This requires the activity of one or more special "error-prone" DNA polymerases; PolV (UmuC,D) is the predominant one in E. coli. The replication errors introduced by the activity of these polymerases are apparently the main basis for UV mutagenesis. Errors may include transitions, transversions, and the insertion or deletion of one to several bases. (If your only options were mutation or death, which would you choose?) Note that the SOS response does not repair the DNA damage; it only buys more times for other repair mechanisms. Components of the SOS pathway are not expressed under normal growth conditions, their production must be induced by damaged DNA. The complex mechanism by which the damage is recognized, and the SOS response proteins (such as Pol V) induced, is beyond the scope of the present discussion.

mutation: basic features of the processbio.classes.ucsc.edu/bio105/winter12/notes/12 13...

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