BIOL 3301 Chapter 19 Gene Mutation and DNA Repair, and Recombination

mutation

• a heritable change in the genetic material

• provide allelic variation

  • foundation for evolutionary change needed for a species to adapt to changes in the environment

  • new ? more likely to be harmful than beneficial to the individual and often are the cause of diseases

evolved

mutations can be quite harmful, therefore organisms have ? mechanisms to repair DNA damage

mutations

• change in chromosome structure

• change in chromosome number

• changes in DNA of a single gene

  • can affect the molecular and phenotypic expression of genes

    • molecular: altered DNA sequence→ diff mRNA→ diff amino acid sequence→ altered phenotype

gene mutations

• molecular changes in the DNA sequence of a gene

point mutation

• a change in a single base pair

  • can involve a base substitution

    • transition: change pyrimidine→ pyrimidine or purine→ purine (more common)

    • transversion: pyrimidine→ purine or purine→ pyrimidine

transition

• change pyrimidine→ pyrimidine

• change purine→ purine

• more common than the other type of base substitution

transversion

• pyrimidine→ purine

• purine→ pyrimidine

silent mutations

• base substitutions that don’t alter the amino acid sequence of the polypeptide

  • due to degeneracy of the genetic code

missense mutations

• those base substitutions in which an amino acid change does occur

• ex., sickle cell disease

• unlike sickle cell disease, a ? mutation may have no detectable effect on protein fx, , and the mutation is said to be neutral (more likely if new amino acid has similar chemistry to the amino acid it replaced)

neutral mutation

• missesnse mutation may have no detectable effect n protein x. more likely to occur if new amino acid has chemistry to the amino acid it replaced

missense, nonsense, silent mutation

• let’s say a transversion mutation occurred int he protein coding portion of a gene. what kind of mutation could this potentially cause?

missense mutation in sickle cell

• ? mutation: changes a single amino acid in a protein

• ? caused by a ? mutation in the beta globin gene

  • mutation changes Glu to Val at position 6

• mutation leads to abnormal Hb, causing RBCs to sickle and affect O2 transport

glutamic acid→ valine

• missense mutation at position 6 of sickle cell disease

• normal beta globin vs sickle cell beta globin

  • originally polar and acidic→ nonpolar

  • NH2- Valine- histidine- leucine-threonine-proline-?- glutamic acid

mutations

• alter the coding sequence within a protein-encoding gene; can have various effects on polypeptide

  • nonsense: base substitutions that change a normal codon to a stop codon

  • frameshift: add/delete a number of nucleotides not divisible by 3

    • shifts the reading frame so that translation of the mRNA results in a completely different amino acid sequence downstream of the mutation

nonsense mutations

• base substitutions that change a normal codon to a stop codon

frameshift mutations

• add/delete a number of nucleotides not divisible by 3

  • shifts the reading frame so that translation of the mRNA results in a completely different amino acid sequence downstream of the mutation

indels

• mutations may also involve the addition or deletion of short sequences of DNA

silent mutation

• base substitution

• no amino acids altered

• prob no effect on protein fx

• mutated mRNA sequence is the same as the coding strand (but U instead of T). template strand is complementary and antiparallel to coding strand.

missense mutation

• base substitution

• 1 amino acid altered

• neutral or inhibitory effect on protein fx

• mutated mRNA sequence is the same as the coding strand (but U instead of T). template strand is complementary and antiparallel to coding strand.

nonsense mutation

• base substitution to change a normal codon→ stop codon

• many amino acids altered

• negative effect on protein fx

• mutated mRNA sequence is the same as the coding strand (but U instead of T). template strand is complementary and antiparallel to coding strand.

frameshift mutation

• addition/deletion

• many amino acids altered

• negative effect on protein fx

• mutated mRNA sequence is the same as the coding strand (but U instead of T). template strand is complementary and antiparallel to coding strand.

wildtype

• the relatively prevalent genotype in a natural population

• genes with multiple alleles may have 2 or more ?

forward mutation

• changes the wild-type genotype into some new variation

reversion mutation

• changes a mutant allele back to the wild-type

deleterious mutations

• decrease the chances of survival

  • most extreme are lethal mutations

beneficial mutations

• enhance the survival or reproductive success of an organism

environment

• the ? can affect whether a given mutation is deleterious or beneficial

conditional

• some mutations are ?

  • they affect phenotype only under a defined set of conditions

  • ex., temperature-sensitive mutation

position effect due to regulatory sequences

• a chromosomal inversion flips a section of DNA

• this move’s gene A’s promoter next to gene B’s regulatory sequence

• since regulatory sequences can work in both direction (bidirectional), gene B’s regulatory elements may now activate gene A

  • regulatory sequences are often bidirectional, so gene A may now show the expression pattern of gene B

• gene A is now expressed abnormally

position effects from translocation to a heterochromatic chromosome

• a ? moves a gene from a euchromatic (open, active) region to a heterochromatic (dense, silent) region

• in heterochromatin (dense, silent) region

• in heterochromatin, DNA is tightly packed and genes are turned off

• effect: the relocated gene becomes

breakpoint

• a chromosomal rearrangement may affect a gene bc the chromosomal ? occurs within the gene

  • site of breaking and rejoining

position effect

• a gene may be left intact after a chromosomal rearrangement, but its expression may be altered bc of its new location

• 2 reasons

  • movement to a position next to regulatory sequences

    • gene A may show expression pattern of gene B

  • movement (translocation) to a heterochromatic region (which is a condensed chromatin and not expressed)

    • gene becomes inactive, even though its sequence is unchanged bc the gene is moved into a silenced region of the genome

germ line cells

• cells that give rise to gametes such as eggs and sperm

somatic cells

• all other non-egg/sperm cells

germ line mutations

• occur directly in a sperm or egg cell, or in one of their precursor cells

• passed on to offspring

• mutation is found in every cell of the body

• half of the gametes in the mature individual may carry the mutation

somatic mutations

• occur directly in a body cell that is not part of the germ-line

• occurs in body cells after fertilization

• not inherited; affects only parts of the body (a “patch”)

• mutation is found in a specific area of the body

• no gametes carry the mutation

germ line mutations

• occur in gametes

• passed on to half of the gametes in the next generation; mutation found in whole body

somatic mutations

• result in patches of affected area

• the size of the patch will depend on the timing of the mutation. the earlier the mutation, the larger the patch

• an individual with ? regions that are genotypically different from the rest of the body is called a genetic mosaic

• mutations not present in gametes

genetic mosaic

• an individual with somatic regions that are genotypically different from the rest of the body

spontaneous mutations

• result from abnormalities in cellular/biological processes

  • e.g., errors in DNA replication

• underlying cause originates within the cell

induced mutations

• caused by environmental agents

• agents that are known to alter DNA structure are termed mutagens

  • these can be chemical or physical agents

spontaneous deamination of cytosine

• removal of an amino group from the cytosine base

  • the other bases are not readily deaminated

  • converts cytosine→ uracil + NH3

  • uracil is not normally found in DNA, so this can be recognized and repaired

• DNA repair enzymes can recognize uracil as an inappropriate base in DNA and remove it

  • however, if the repair system fails, a C-G to A-T mutation will result during subsequent rounds of DNA replication

    • C-G base pair becomes an A-T base pair → permanent mutation

spontaneous deamination of 5 methylcytosine

• 5 methylcytosine can be deaminated into thymine, a normal constituent of DNA

  • 5 methylcytosine→ thymine + NH3

  • thymine is a normal DNA base, so this mutation is harder to detect and repair may lead to permanent base changes

• repair enzymes cannot determine which of the 2 bases on the 2 DNA strands is the incorrect base

• for this reason, methylated cytosine bases tend to create hot spots for mutation

oxidative stress

• an imbalance btw the production of reactive oxygen species (ROS) and an organisms’s ability to break them down

  • ROS: hydrogen peroxide, superoxide, hydroxyl radical

• may lead to DNA damage and mutation

  • ROS over accumulation can lead to oxidative DNA damage

  • ex., guanine → 7, 8-dihydro 7-oxoguanine (8-oxoG)

    • 8-oxoG mistakenly pairs with adenine during replication (normal G pairs with C)

      • the original G-C base pair becomes a T-A base pair→ permanent point mutation

reactive oxygen species ROS

• aerobic organisms produce ? which include

  • hydrogen peroxide

  • superoxide

  • hydroxyl radical

• body tries to block this buildup (bc accumulation can lead to oxidative DNA damage)

  • enzymes

    • superoxide dismutase and catalase

  • antioxidants

oxidative DNA damage

• results from reactive oxygen species ROS overaccumulation

  • hydrogen peroxide, superoxide, hydroxyl radical

    • body enzymes like superoxide dismutase and catalase, and antioxidants don’t break enough ROS down

• ex., guanine → 7, 8-dihydro 7-oxoguanine (8-oxoG)

  • 8-oxoG mistakenly pairs with adenine during replication (normal G pairs with C)

    • the original G-C base pair becomes a T-A base pair→ permanent point mutation

trinucleotide repeat expansion

• several human genetic diseases are caused by an unusual form of mutation

• e.g., huntington’s disease (autosomal dominant, lethal, no cure)

• certain regions of the chromosome have short sequences repeated in tandem

  • in unaffected individuals, these repeats are stable and passed on wo mutation

  • in affected individuals, the length of? has increased above a certain critical size

    • disease symptoms occur

• in some cases, the expansion is within the coding sequence of the gene

• typically, the ? is CAG (glutamine)

  • therefore, the coded protein will contain long tracks of glutamine

    • this causes the proteins to aggregate with each other

    • this aggregation is correlated with the progression of the disease, but may not cause disease symptoms

• some ? disorders progressively worsen in future generations

  • may depends on which parent the mutant allele comes from

    • in huntington disease, the ? is more likely to occur if inherited from the male parent

    • in myotonic muscular dystrophy, the ? is more likely to occur if inherited from the female parent

      • suggests that ? can occur more frequently during oogenesis or spermatogenesis, depending on the gene involved

• these changes can occur during gamete formation

  • offspring will have very different numbers of repeats

• can also increase in somatic cells as a person ages

  • this can increase severity of the disease over time

huntingtons’s disease

• trinucleotide repeat more likely to expand if inherited from the father

• suggests TRNEs can occur more frequently during oogenesis or spermatogenesis, depending on the gene involved

myotonic muscular dystrophy

• trinucleotide repeat more likely to expand if inherited from the mother

• suggests TRNEs can occur more frequently during oogenesis or spermatogenesis, depending on the gene involved

trinucleotide repeat expansion TRNE mechanism

• ? can expand during DNA replication

• trinucleotide repeat sequences (e.g., CTG, CAG) are present in the DNA

• these repeats can form hairpin loops due to C-G base pairing

• during DNA replication

  • DNA polymerase replicates thru the repeat region

  • a hairpin forms in the new (daughter) strand

• hairpin causes DNA polymerase to slip off

• when DNA polymerase resumes replication, it recopies the repeat region

• this results in a longer repeat region in the daughter strand

• DNA gap repair seals the new strand, locking in the extra repeats

• TRNE expands, over time this leads to disease-causing mutations

mutagens

• agents that alter the DNA structure and thereby cause mutations

• type of induced mutations

• 2 primary concerns

  • ? often involved in the development of human cancers

  • ? can cause gene mutations that may have harmful effects in future generations

• an enormous array of agents can act as ?

• usually classified as chemical or physical

chemical mutagens

• 3 main types

  • base modifiers

    • some covalently modify base structure

    • others disrupt pairing by alkylating bases

  • intercalating agents

    • directly interfere with replication process

    • slip btw DNA base pairs, distorting DNA structure, which interferes with DNA replication

      • can cause insertions or deletions

  • base analogues

    • incorporate into DNA and disrupt structure and normal base pairing

    • look like normal bases, but aren’t. can be mistakenly used by cell and inserted into DNA during replication

• mutagens

  • agents that alter the DNA structure and thereby cause mutations

  • type of induced mutations

  • 2 primary concerns

    • ? often involved in the development of human cancers

    • ? can cause gene mutations that may have harmful effects in future generations

• tldr

  • chemical that react with bases change their structure and make them pair weird

physical mutagens

• often cause breaks or abnormal bonds in DNA

• include radiation

  • X rays, gamma rays, ionizing radiation, UV light

• agents that alter the DNA structure and thereby cause mutations

• type of induced mutations

• 2 primary concerns

  • ? often involved in the development of human cancers

  • ? can cause gene mutations that may have harmful effects in future generations

nitrous acid

• a type of CHEMICAL MUTAGEN

• chemically reacts with DNA bases

• causes deamination (removal of an amino group) of:

  • cytosine→ uracil

    • U pairs with A during replication

    • this causes a C-G to a T-A mutation over time

  • adenine→ hypoxanthine

    • hypoxanthine pairs w/ cytosine

    • this causes an A-T to G-C mutation over time

• base deamination by ? leads to incorrect base pairing, which results in point mutations after DNA replication

base analogues

• become incorporated into daughter strands during DNA replication

• look like normal bases, but aren’t

• ex., 5 bromouracil is a thymine analogue

  • it can be incorporated into DNA instead of thymine

  • it can pair with guanine or adenine

    • keto form→ pairs with adenine (normal)

    • enol form→ pairs with guanine (abnormal)

• chemical mutagen that increase the chance of transition mutations (purine-purine or pyrimidine-pyrimidine switch)

5 bromouracil 5BU

• 5BU is a base analogue (looks like thymine)

• can exists in 2 forms

  • keto form→ pairs with A (normal)

  • enol form→ pairs with G (abnormal)

• 5BU incorporated into DNA instead of thymine

• if 5BU switches to enol form, it mispairs with G

• after 1 round of replication, G is in place of A

• after another round, a G-C pair replaces original A-T

• result: a permanent point mutation (A-T → G-C)

• a chemical mutagen that increases the chance of transition mutations (purine-purine or pyrimidine-pyrimidine switch)

ionizing radiation

• a type of physical mutagen

  • ex., X rays, gamma rays

    • short wavelength

    • high energy

    • can penetrate deeply into biological molecules

    • creates chemically reactive molecules (FREE RADICALS)

  • can cause

    • base deletions

    • oxidized bases

    • single nicks in DNA strands

    • cross linking

    • chromosomal breaks

nonionizing radiation

• type of physical mutagen

  • ex., UV light

    • has less energy

    • cannot penetrate deeply into biological molecules

    • causes the formation of cross-linked thymine dimers

      • thymine dimers form when UV light hits DNA

      • 2 adjacent thymine bases on same DNA strand that covalently bond> distort DNA double helix (bulky, abnormal structure)

    • thymine dimers may cause mutations when that DNA strand is replicated

      • if not repaired by nucleotide excision repair

mutation rate

• the likelihood that a gene will be altered by a new mutation

• commonly expressed as the # of new mutations in a given gene per cell generation

  • how likely a gene is to be altered by a new mutation.

  • number of new mutations per cell generation.

• range of 10^-5 to 10^-9 per generation

  • humans: each generation adds about 100-200 new mutations to the genome (100-200 mutations/generation)

• ? for a given gene isn’t constant

  • can be increased by presence of mutagens

• vary substantially btw species and even within different strains of the same species

DNA repair

• vital to the survival of all organisms bc most mutations are deleterious

• living cells contain several ? systems that can fix different type of DNA alterations

  • direct repair

  • base excision repair and nucleotide excision repair

  • mismatch repair

  • homologous recombination

  • nonhomologous end joining (for double stranded breaks!)

• in most cases, ? is a multistep process

  • an irregularity in DNA structure is detected

  • the abnormal DNA is removed

  • normal DNA is synthesized

direct repair

• enzyme recognizes an incorrect alteration in DNA structure and directly converts the structure back to the correct form

  • specific enzymes can reverse the covalent modifications of nucleotides

    • photolyase

      • repair thymine dimers

      • splits the dimers restoring the DNA to its original condition

      • uses energy of visible light for photoreactivation

    • alkyltransferase

      • repairs alkylated bases

        • transfers the methyl or ethyl group from the base to a cysteine side chain within the alkyltranferase protein

        • surprisingly, this permanently inactivates alkyltransferase

photolyase

• enzyme that performs direct repair of a thymine dimer

  • thymine dimers form when UV light causes 2 adjacent thymines to bond

• uses visible light energy to break the bonds btw the thymines (photoreactivation)

  • splits the dimers

• restore DNA to normal structure (2 separate thymines)

alkyltransferase

• performs direct repair of an alkylated base

  • sometimes guanine is incorrectly modified into O⁶-methylguanine (a mutagenic, alkylated base)

• repairs alkylated bases

  • removes the methyl (or ethyl) group from the damaged base

  • transfers the group to a cysteine side chain on itself

• this repair action permanently inactivates the ? enzyme

  • the enzyme can only be used once (“suicide enzyme”)

base excision repair BER

• removes a damaged base, a segment of DNA in this region is excised, then the complementary DNA strand is used as a template to synthesize a normal DNA strand

• involves DNA N-glycosylases enzyme category

  • recognize an abnormal base and cleave the bond btw it and the sugar in DNA

• depending on the species, this repair system can eliminate abnormal bases like

  • uracil; 3-methyladenine; 7-methylguanine

base excision repair BER

• DNA N-glycosylases detects and removes the abnormal base

  • removes an abnormal base and cleaves the bond btw the base and the sugar

  • leaves an AP site (apurinic/apyrimidinic site) (missing purine or pyrimidine)

• AP endonuclease cuts the DNA backbone at the 5’ side of the missing base

• DNA polymerase

  • in E.coli:

    • DNA pol I 5’ → 3’ exonuclease removes damaged section and fills in correct nucleotides, DNA ligase seals the region

  • in humans

    • Pol β can remove and replace just one nucleotide. DNA ligase seals.

    • Or Pol δ/ε synthesize a new strand (flap formed).

      • flap is removed by flap endonuclease. DNA ligase seals the region

nucleotide excision repair NER

• several nucleotides in the damaged strand are removed from the DNA and the undamaged strand is used as a template to resynthesize a normal strand

• can repair many types of DNA damage

  • thymine dimers and chemically modified bases

  • missing bases

  • some types of crosslinks

• found in all eukaryotes and prokaryotes

• however, its molecular mechanism is better understood in prokaryotes

  • in E coli

    • 4 proteins required

      • UvrA, UvrB, UvrC, UvrD

        • involve in UltraViolet light Repair of pyrimidine dimers

          • also important in repairing chemically damaged DNA

      • UvrA, B, C, and D recognize and remove a short segment of damaged DNA

      • DNA polymerase and ligase finish the repair job

  • in humans

    • several human diseases have been showed to involve inherited defects in genes involved in ?

      • xeroderma pigmentosum (XP)

        • can be caused by defects in 7 different ? genes

      • cockayne syndrome CS

        • increased sensitivity to sunlight is a common characteristic

nucleotide excision repair in ecoli

• several nucleotides in the damaged strand are removed from the DNA and the undamaged strand is used as a template to resynthesize a normal strand

• can repair many types of DNA damage

  • thymine dimers and chemically modified bases

  • missing bases

  • some types of crosslinks

• proteins required

  • UvrA, UvrB, UvrC, UvrD

    • involve in UltraViolet light Repair of pyrimidine dimers

      • also important in repairing chemically damaged DNA

  • UvrA, B, C, and D recognize and remove a short segment of damaged DNA

  • DNA polymerase and ligase finish the repair job

base mismatch

• another abnormality in DNA

• structure of the DNA double helix obeys the AT/GC rule of base pairing

  • however, during DNA replication, an incorrect base may be added to the growing strand by mistake

• DNA polymerases have a 3’ to 5’ proofreading ability that can detect base mismatches and fix them

• if proofreading fails, the ? repair system comes to the rescue

  • MutS protein slides along DNA and finds a mismatch

  • MutS/MutL complex binds to MutH, which is already bound to a hemimethylated sequence

• ? repair systems are found in all species

  • important: these systems are specific to the newly made strand

  • molecular mechanism of ? repair in Ecoli

    • 3 proteins: MutL, MutH, MutS

      • detect mismatch and direct its removal from the newly made strand

      • proteins are “Mut” bc their absence leads to a much higher mutation rate than normal

• MutH can distinguish btw the parental vs daughter strand

  • prior to replication, both parental strands are methylated

  • immediately after replication, the parental strand is methylated, whereas the newly made daughter strand is not

mismatch repair

• wrong base is paired during DNA replication

• normally, DNA pol 3’ → 5’ exonuclease can proofread and fix mistakes, but if it misses one, ? comes to rescue

• in E. coli

  • MutS scans the DNA and detects the msimatch

  • MutL joins MutS to form a complex

  • MutH already bound near the mismatch at a hemimethylated site (where only the parent strand is methylated)

  • MutH cuts the new (unmethylated) strand at the mistake site

  • MutU unwinds the DNA

  • an exonuclease removes a section of the strand, including the mismatch

  • DNA pol adds the correct nucleotides

  • DNA ligase seals the gap

• note

  • MMR occurs after replication to fix remaining errors

  • only the newly made strand is corrected (recognized by lack of methylation)

  • proteins involved are termed Mut bc their absence leads to a much higher mutation rate than normal

DNA double strand breaks

• VERY DANGEROUS

• breakage of chromosome into pieces

• caused by ionizing radiation and chemical mutagens

  • also caused by reactive oxygen species which are the byproducts of cellular metabolism

  • 10-100 breaks occur each day in a typical human cell

• breaks can cause chromosomal rearrangements and deficiencies

• they may be repaired by

  • homologous recombination repair HRR

  • nonhomologous end joining NHEJ

nonhomologous end joining

• broken ends are recognized by end-binding proteins

  • formation of crossbridge

• processing may result in deletion of a small region

  • not error free

• fixes dangerous double stranded breaks in DNA (both strands are broken)

  • doesn’t need a template (unlike homologous recombination repair- other method to repair double stranded breaks)

• steps

  • special proteins bind broken ends of DNA

  • proteins from crossbridge to keep ends close together

  • extra/damaged DNA is trimmed; more proteins recruited

  • DNA pol fills in missing bases, DNA ligase seals the break

    • fast, but error prone

    • used in non-dividing cells or when no template available