CHAPTER 16 GENE MUTATION AND DNA REPAIR

Why mutagens interest?

1. Mutagens are often involved in the development of human cancers 2. Mutagens can cause gene mutations that may have harmful effects in future generations

Transition

A base substitution of a pyrimidine to another pyrimidine (C > T) or a purine to another purine (A > G) Purines are AG Pyrimidines are CUT

transversion

A base substitution where a purine is interchanged with a pyrimidine

AP endonuclease

A endonuclease that recognizes apurinic or apyrimidinic sites (AP) and makes a nick in the DNA backbone on the 5' side of the AP site. AP site is created by DNA N-glycosylase. Functions in Base Excsision Repair

Base Substitution

A point mutation in which one base is substituted for another. Ex. transition or transversion

Point Mutatoin

A point mutation is a change in a single base pair within the DNA. (base substitution, deletion or addition)

mutagens

Agents that are known to alter the structure of DNA. Mutagens can be chemical substances or physical agents that react with DNA

lethal mutation

An extreme example of a deleterious mutation where death is the outcome

Chemical mutagen types

Base modifiers, intercalating agents and base analogues

Mismatch Repair Sytem

DNA polymerases (3-5 proofreading ability) and methyl-directed mismatch repair can detect and correct base pair mismatch

Common DNA Repair Systems

Direct Repair, Base excision and nucleotide excision repair, mismatch repair, homologous recombination repair, non-homologous end joining

Nonhomologous End Joining (NHEJ)

Double-strand breaks in the DNA double helix are caused by ionizing radiation, chemical mutagens and free radicals. Double-strand breaks can lead to chromosomal mutations. The nonhomologous end joining (NHEJ) pathway joins broken DNA ends together. 1) The DSB is recognized by end-binding proteins 2) End-binding proteins then recognize additional proteins that form a cross-bridge that prevents the two ends from drifting apart. 3) Additional proteins are recruited to the region that process the ends of the broken chromosome by digestion of particular DNA strands-small amount of genetic material may be deleted. 4) Gaps are filled by DNA polymerase and ends ligated together by DNA ligase.

Photylase

For example, yeast cells, bacteria, plants and some animals can repair thymine dimers using an enzyme called photolyase that can split the thymine dimer to restore DNA to its original condition This enzyme is flavoprotein that contains two light-sensitive cofactors that require light to directly restore the structure of DNA.

Direct Repair

In a few cases, the covalent modifications of nucleotides can be reversed by specific enzymes Includes Photlyase and O6-alkylguanine alkyltransferase

Nonionizing Radiation

Include UV light. Has less energy and cannot penetrate deeply into biological molecules. Causes formation of cross-linked thymine dimers which can cause mutations when that DNA strand is replicated.

MutL

Mut L forms a complex with Muts protein in the base mismatch repair system to bind to mutH on the GATC site (site where methylation occurs on adenine).

Position Effect

Position effect occurs when a phenotypic change is observed when a translocation or inversion occurs in one of two ways: Gene is moved next to the regulatory sequence of another gene such that it is regulated in a different manner Gene is moved in a section that is compacted differently thus is transcriptional active to a different degree

UvrA

Protein in Nucleotide Excision Repair (Ultraviolet light repair of pyrimidine dimers). Forms a complex with UvrB (UvrA/UvrB complex) that tracks along the DNA in search of damaged DNA (thymine dimer or modified bases). Releases after damage is detected.

Reactive Oxygen species

ROS generated within cells can chemically modify bases (usually G), causing mispairing (oxidized G will pair with A leading to one strand to be TA bp-a transversion). Antioxidants and other enzymes in cells help prevent the buildup of these ROS-needed in certain amounts in the immune response against pathogens and cell signaling

Breakpoint mutations

The gene sequence can also be altered at the breakpoint of inversions and translocations. The breakpoints may affect the coding sequence of a gene so that the coding sequence is incorrect or these breakpoint may affect the regulatory regions of a gene so that the coding sequence is now transcribed incorrectly.

Mutation frequency

The mutation frequency is the number of mutant genes divided by the total number of genes within the population. The mutation frequency depends not only on the mutation rate, but also on the Timing of the mutation during population growth Likelihood that the mutation will be passed on to future generations

Nucleotide Excision Repair diseases

These include xeroderma pigmentosum (XP), Cockayne syndrome (CS) and PIBIDS A common characteristic of both syndromes is an increased sensitivity to sunlight

Physical Mutagens Types

X-rays and UV light

MutU

a helicase protein in the base mismatch repair system that separates the strands at the GATC cleavage site (methylation occurs on adenine)

Silent mutation

a mutation on the protein coding sequence that do not alter the amino acid sequence; most likely to occur on the third base; also a neutral mutation

Mutations described by effects on phenotype of function

beneficial, deleterious, null mutations, gain-of-functions, conditional

O6-alkylguanine alkyltransferase

can remove the methyl or ethyl groups from guanine bases that have been mutagenized by agents such as nitrogen mustards and ethyl methanesulfonate. During the process, the alkyltransferase is permanently inactivated so it can only be used once because the alkyl group is transferred from the guanine to a cysteine side chain within the protein.

DNA N-glycosylase

category of enzymes that is involved in Base Excision Repair. Can recognize an abnormal base-like uracil, thymine dimers, 3-methyladenine, 7-methylguanine-and cleave bond between it and sugar in DNA

null mutation

cause loss of all function of the protein encoded by the gene, and are so give a phenotype corresponding to removal of the gene (usually recessive)

reverse mutation

changes a mutant genotype back to the wild-type genotype

forward mutation

changes the wild-type genotype into some new variation-a mutant allele if it is rare in the population

nitrous acid

deaminates amino groups with keto groups causing cytosine to change to uracil and adenine to hypoxanthine; don't pair appropriately, UA/HC

mutation rate

is the likelihood that a gene will be altered by a new mutation Commonly expressed as the number of new mutations in a given gene per generation It is in the range of 10-5 to 10-9 per generation and is not constant. Can increase or decrease by the presence of mutagens.

5' UTR/3' UTR

may alter the ability of mRNA to be translated, may alter mRNA stability

Splice recognition sequence

may alter the ability of pre-mRNA to be properly spliced

regulatory element/operator site mutation effect

may disrupt the ability of a gene to be properly regulated

Promoter mutation effect

may increase or decrease the rate of transcription

conditional mutations

mutation that affects the phenotype only under a defined set of conditions. Ex. temperature-sensitive mutation

MutS

protein in mismatch repair system that detects base mismatch. Then forms a complex with MutL

MutH

protein in the base mismatch repair system. MutH, which can distinguish parental and daughter strand because parental is methylated, is located on daughter/nonmethylated strand and makes a cut in it

Protein coding region

region of a gene whose sequences will be translated into protein

regulatory regions

region on a gene like a promoter or other regulatory sequences that impact how or if a gene is transcribed.

untranslated regions

region on a gene that is transcribed but not translated

Spontaneous mutations

result from cellular/biological processes-errors in DNA replication, recombination, transposable elements, changes in bases, metabolic reactive oxygen species originates within the cell and arise by chemical changes that happen spontaneously to bases TYPES: depurination, deamination, tautomeric shift, oxidation by reactive oxygen species

transcribed region

sequences on a gene that will be on the primary, unprocessed transcript (containing introns

Base Analogues

similar to normal bases but undergo tautomeric shifts more frequently Include 5-bromouracil and 2-aminopurine

Intercalating Agents

stack in the double helix and distort, cause single base deletions or additions Include proflavin

Random Mutation Theory

states that mutations are a random process-they can occur in any gene and do not involve exposure of an organism to a particular condition that causes specific types of mutations to happen. In the cases where random mutations provide a mutant organism with an advantage, such occur as a matter of change, growth conditions may select for organisms that happen to carry them preferentially.

Tautomeric Shifts

tautomers are bases that exist in two forms by the shift of a hydrogen atom and a switch of a single bond and an adjacent double bond. o Keto form of guanine and thymine is more stable than the enol form. o Amino form of adenine and cytosine is more stable than the imino form. o If one of the bases in in enol or imino form, it will not base pair AT/GC but TG/CA. With a tautomeric shift, there is still always base-pairing between a purine and pyrimidine, just the opposite pairs than normal For a mutation to occur it must shift immediately prior to DNA replication where one strand will be incorrect. This can be repaired but failures are possible.

Depurination

the covalent bond between a purine base can break, creating an apurinic site (depurination). The covalent bond between deoxyribose and a purine base is somewhat unstable and occasionally undergoes a spontaneous reaction with water that releases the base from the sugar. The most common naturally occurring spontaneous mutation. Rate of mutation is higher if DNA is exposed to agents that cause modifications such as alkyl addition. If this occurs just prior to DNA replication, it may cause a mutation.If repair systems fail, DNA replication can lead to one strand can have any of the four bases in the apurinic site.

gain-of-function mutation

usually dominant mutations that increase the amount of add to the function of a gene's protein, giving an altered phenotype

Suppressor Mutations

A second mutation, at a new location, will sometimes affect the phenotypic expression of a first mutation, reverting the phenotype of the individual from mutant back to wild-type (or close to wild-type). ). This term refers to the fact that a suppressor mutation acts to suppress the phenotypic effects of another mutation. Two Types: intragenic and intergenic

Deamination of Cytosine

A spontaneous mutation that occursr on cytosine bases-others are not readily deaminated-that removes the amino group and produces uracil. -The uracil can be recognized and repaired, if not then during DNA replication the uracil will hydrogen bond with adenine instead of the guanine that the deaminated cytosine would have bonded to.

Base Excision Repair

Can work on uracil, 3-methyladenine, 7-methylguanine and pyrimidine dimers, depending on the species BER involves DNA N-glycosylase enzymes that can recognize abnormal bases and cleave the bond between it and the sugar in DNA. AP Endonuclease recognizes missing base and cleaves DNA backbone on 5' side of missing base DNA polymerase uses 5 to 3 exonuclease to remove damaged region and then fills with normal DNA. DNA ligase seals the region

Homologous Recombination Repair (HRR)

Double-strand breaks in the DNA double helix are caused by ionizing radiation, chemical mutagens and free radicals. Double-strand breaks can lead to chromosomal mutations. The homologous recombination repair (HRR) pathway uses the broken chromosome's sister chromatid, or occasionally it's homologous chromosome as the template for repair. 1) The DSB is processed by a short digestion of DNA strands at the break site. 2) Exchange of DNA strands between the broken and unbroken sister chromatids. 3) The unbroken strands are then used as templates to synthesize DNA in the region where the break occurred. 4) Crisscrossed strands are resolved, which means they are broken and then rejoined in a way that produces separate chromatids. Because sister chromatids are genetically identical, an advantage is that it is error-free. However sister chromatids are only available during the S and G2 phase of the cell cycle in eukaryotes.

Translesion DNA Polymerases

If DNA damage that distorts the DNA is not repaired prior to replication, DNA Pol III will not replicate through the lesion. However, error-prone translesion DNA polymerases can use the aberrant DNA as a template. Regions of DNA replicated by translesion polymerases are much more likely to contain mutations because of the lower fidelity of DNA replication by these polymerases.

Deamination of methylcytosine

If deamination occurs on a methylcytosine, it creates thymine, making it difficult for DNA repair enzymes to distinguish the correct base from an altered base. -Since thymine is a regular constituent of DNA, DNA repair proteins cannot distinguish which is the incorrect base-the thymine that was produced during deamination or the guanine in the opposite strand that originally base-paired with the methylated cytosine. Methylated cytosines are hot spots for mutations.

Nucleotide Excision Repair

Important and widespread DNA repair mechanism in pro and eukaryotes for thymine dimers; modified, missing or cross-linked bases using UvrA-D proteins (ultraviolet light repair of pyrimidine dimers) 1) UvrA/UvrB complex track along DNA and detects damage 2) Uvr A is released and UvrC binds 3) UvrC makes cut on both side of thymine dimer 4) UvrD (helicase) removes the damaged region, UvrB and UvrC 5) DNA polymerase fills in gap and DNA ligase seals

methyl-directed mismatch repair

In E. Coli, three proteins, MutL, MutH and MutS detect the mismatch and direct its removal from the newly made strand. 1) MutS locates base pair mismatch 2) MutS/MutL complex forms and interacts with MutH by looping mechanism 3) MutH, which can distinguish parental and daughter strand because parental is methylated, is located on daughter/nonmethylated strand and makes a cut in it 4) MutU, a helicase, separates strands at cleavage site 5) Exonuclease digests the nonmethylated strand beyond base mismatch 6) Gap is filled by DNA polymerase and joined by DNA ligase.

Ionizing Radiation

Includes x-ray and gamma rays. Have short wavelength and high energy can penetrate deeply into biological molecules. Creates chemically reactive free radicals. Can cause base deletions, single nicks in DNA Strands, cross-linking, chromosomal breaks

Induced mutations

Induced mutations are caused by environmental agents either chemical or physical. Originates outside of the cell

UvrC

Protein in Nucleotide Excision Repair (Ultraviolet light repair of pyrimidine dimers). Forms a complex after damaged has been detected by UvrA/UvrB complex and UvrA has released (UvrB/UvrC). UvrC makes cuts on both sides of the damaged site-typically eight nucleotides from the 5' end and five away from the 3' end of the site.

UvrB

Protein in Nucleotide Excision Repair (Ultraviolet light repair of pyrimidine dimers). Forms a complex with UvrA (UvrA/UvrB complex) that tracks along the DNA in search of damaged DNA (thymine dimer or modified bases) and remains attached until damaged region is removed.

UvrD

Protein in Nucleotide Excision Repair (Ultraviolet light repair of pyrimidine dimers). Uvrd, which is a helicase, removes the damaged region along with UvrB and UvrC.

Mutation Effects

The occurrence of a mutation within the coding sequence of a structural gene can have a variety of effects on the amino acid sequence of the polypeptide that is encoded by a gene, like the following: none, silent, missense, nonsense, frameshift

Ames Test

The test uses a strain of Salmonella typhimurium that cannot synthesize the amino acid histidine It has a point mutation in a gene involved in histidine biosynthesis A second mutation (i.e., a reversion) may occur restoring the ability to synthesize histidine The Ames test monitors the rate at which this second mutation occurs

Intragenic suppressors

a second mutant site, within the same gene as the first mutation, that acts to suppress the phenotypic effects of the first mutation site. Often involves a change in the protein structure that compensates for an abnormality in protein structure caused by the first mutation to restore protein function to wild type

Frameshift

a mutation on the protein coding sequence that involves the addition or deletion of a number of nucleotides not divisible by three; results in a completely different amino acid sequence downstream of a muation

Neutral mutation

a mutation on the protein coding sequence when a mutation has no detectable effect on protein function. Mutation less likely to be neutral by size, location and shift.

Missense mutation

a mutation on the protein coding sequence where amino acid change does occur; can be neutral if single and similar chemistry and shape

Nonsense mutation

a mutation on the protein coding sequence. a change from a normal codon to a stop codon producing a truncated polypeptide

Down promoter mutation

a mutation outside of a gene's coding sequence that decreases the rate of transcription because it is less like the consensus sequence

Up promoter mutation

a mutation outside of a gene's coding sequence that increases the rate of transcription because it is more like the consensus sequence

Intergenic suppressors

a second mutation in a different gene from the first mutation that compensates for the effects caused by the first mutation. (AKA extragenic) Usually work by changing the expression of one gene that compensates for a loss-of-function mutation affecting another gene OR slightly alters the function of second gene so that it is like the first gene OR a mutation in a second gene allows it to continue interacting with a mutant first gene to act as a dimer and continue function OR mutation inactivates/activates function of downstream gene in regulatory pathway can identify genes in the same biochemical pathway

5-bromouracil

a thymine analogue that base-pairs with adenine; a high frequency Tautomeric shift of 5BU base pairs with guanine, therefore a AT base pair changes to A-5BU, in one strand in replication G-5BU and finally one strand is GC

Base modifiers

chemically alter the bases, change base pairing Include nitrous acid, nitrogen mustard, and ethyl methanesulfonate

alkylating agents

methyl or ethyl groups are covalently attached to bases; ex nitrogen mustard gas and ethyl methanesulfonate; disrupts appropriate base pairing between nucleotides

True reversion

when a mutation revers the genotypic sequence back to wild-type

wild-type

wild-type refers to the most common genotype in a population

deleterious mutation

will decrease the chances of survival or reproductive success of an organism (fitness)

beneficial mutation

will increase the chances of survival or reproductive success of an organism (fitness)