Genetics Chapter 16

proto-oncogenes

code for proteins that help to regulate cell growth and differentiation. are often involved in signal transduction and execution of mitogenic signals, usually through their protein products Upon ''activation'', it (or its product) becomes a tumor-inducing agent, an oncogene. include RAS, WNT, MYC, ERK, and TRK

Uracil-DNA glycosylase

removes uracil from DNA. Uracil residues, which result from the spontaneous deamination of cytosine, can lead to a C-to-T transition if unrepaired

transletion in both prokaryotes and eukaryotes

replication blocks can be bypassed by the insertion of nonspecific bases.

Frameshift mutations

result from insertions or deletions of a base pair Can be very severe, especially if they occur early in the coding sequence Except multiples of three, which would reestablish the initial frame of reading

Replication slippage causes repeat expansion

slip happened and now the part of the template is now repeated twice in the daughter strand

Two major processes are responsible for genetic variation

-Mutation - Recombination

spontaneous lesions

1) Depurination 2)Deamination: 3)Oxidatively damaged bases

homology dependent repair systems

1) base excision repair 2) Nucleotide excision repair

In adult cells that have stopped dividing, what types of repair systems are possible?

There are many repair systems that are available: direct reversal, excision repair, transcription-coupled repair, and nonhomologous end joining.

Why are many chemicals that test positive by the Ames test also classified as carcinogens?

There is a very strong correlation between mutagens and carcinogens. As discussed in Problem 14, cancer is a genetic disease. Therefore, any chemical classified as a mutagen by the Ames test should also be considered a carcinogen.

Translesion or bypass polymerases differ from the main replicative polymerases in several ways

They can tolerate unusually large adducts on the bases Have much higher error rate (lack 3' to 5' proofreading activity) Can only add few nucleotides before falling off

Mutations in tumor-suppressor genes

that promote tumor formation are loss-of-function recessive mutations. Causes the encoded gene products to lose much or all of their activity. Cancer to develop, mutation must be present in both alleles of the genes The products of tumor-suppressor genes normally regulate cell-cycle checkpoints and initiate the process of apoptosis When tumor-suppressor genes are mutated or inactivated, cells are unable to respond normally to cell-cycle checkpoints or are unable to undergo apoptosis if DNA damage is extensive Leads to more mutations and development of cancer

transletion in E. Coli

this process requires the activation of the SOS system.

the ames test

uses a number of different strains of Salmonella typhimurium selected for their ability to reveal the presence of specific types of mutations is used extensively during the development of industrial and pharmaceutical chemical compounds

Mechanisms of spontaneous mutations

1) error in DNA replication During DNA replication, DNA polymerase occasionally inserts incorrect nucleotides Although DNA polymerases can proofread and correct these errors, they may persist if not detected and corrected These types of errors, due to mispairing, predominantly lead to point mutations 2) tautomeric shifts can change the bonding structure, allowing non-complementary base pairing May lead to permanent base-pair changes and mutations 3) error in DNA replication Can also lead to frameshift mutations If a DNA strand loops out and becomes displaced small insertions and deletions can occur (indel mutations) Replication slippage can occur anywhere in the DNA but is more common in repeat sequences Hot spots for hereditary diseases Fragile-X, Huntington Disease

types of gene mutations

1) spontaneous mutations 2) induced mutations

Steps of NER

1)Recognition of damaged bases 2)Assembly of a multiprotein complex at the site 3)Cutting of the damaged strand several nucleotides upstream and downstream of the damage site and removal of the nucleotides (~30) between the cuts 4)Use of the undamaged strand as a template for DNA polymerase followed by strand ligation

mismatch repair systems

1)Recognize mismatched base pairs 2)Determine which base in the mismatch is the incorrect one 3)Excise the incorrect base and carry out repair synthesis

SDSA steps

1)Trimming of the 5' ends by an endonuclease 2)Rad51 forms long filaments as it associates with the exposed single stranded region 3)Rad51-DNA filament then search for the undamaged sister for strand invasion 4)The 3' end of the invading strand displaces one of the undamaged sister chromatids, which forms a D-loop (for displacement), and primes DNA synthesis from its free 3' end

Base excision repair steps

1. DNA glycosylases: cleave base-sugar bonds, thereby liberating the altered bases & generating apurinic or apyrimidinic (AP) sites 2. AP endonuclease: nicks the damaged strand upstream of the AP site 3. Deoxyribophophodiesterase (dRpase): removes stretch of neighboring sugar-phosphate residues 4. DNA polymerase: fill up the gap with nucleotides 5. DNA ligase: seals the new nucleotide into the backbone

trinucleotide repeat disease

A common mechanism responsible for a number of genetic diseases is the expansion of a three-base-pair repeat: trinucleotide repeat diseases e.g. human disease called fragile X syndrome - is the most common form of inherited mental retardation, occurring in close to 1 of 1500 males and 1 of 2500 females. - It is manifested cytologically by a fragile site in the X chromosome that results in breaks in vitro.

Base Damage

A large number of mutagens damage one or more bases in DNA UV light Ionizing radiation Result in a replication block

The ras oncogene is continuously active

A single base pair substitution that converts Glycine to Valine at amino acid number 12 of the Ras protein, creates the oncoprotein found in human bladder cancer The missense mutation in the ras oncogene produces an oncoprotein that always bind GTP, even in the absence of normal signals. As a result, Ras oncoprotein continuously propagates a signal that promotes cell proliferation. Mutations that convert the ras proto-oncogene to an oncogene freeze the ras protein into its active conformation, constantly stimulating the cell to divide

Base insertions or deletions (indel mutation)

Addition or deletion of a single base pair or multiple base pairs

Base-excision repair

After DNA proofreading by DNA polymerase, this is the most important mechanism used to remove incorrect or damaged bases The main target is nonbulky damaged bases- result from variety of causes: methylation, deamination, oxidation or the spontaneous loss of a DNA base

two pathways continued

After lesion recognition, the GG-NER and TC-NER pathways utilize largely the same proteins to remove and repair the damaged DNA. The role of both XPC-RAD23B and CSA/CSB is to attract the multiprotein TFIIH complex. Step 3: the XPD and XPB helicases unwind DNA helix around the lesion After removal of the damaged base and surrounding DNA, the gap is filled by a DNA polymerase assisted by the RFC and PCNA proteins. The last step of NER involves ligation of the new strand to the surrounding DNA by one of two ligation complexes (XRCC1/LIG3 or FEN1/LIG1).

Nucleotide excision repair

Can correct bulky adducts and correct damage to more than one base Relieves stalled replication forks and transcription complexes Recognizes and corrects DNA lesions due largely to UV damage Relieves stalled replication forks and transcription complexes Interestingly, two autosomal recessive diseases in humans, xeroderma pigmentosum (XP) and Cockayne syndrome, are caused by defects in nucleotide-excision repair.

UV light

Cause damage to nucleotide bases in most organisms Generate photoproducts: two different lesions that unite adjacent pyrimidine residues in the same strand (a)Cyclobutane pyrimidine dimer (b)6-4 photoproduct

mismatch repair

Corrects errors in replication that are not corrected by the proofreading function of the replicative DNA polymerase. Repair is restricted to the newly synthesized strand, which is recognized by the repair machinery in prokaryotes because it lacks methylation marker

Direct reversal of damaged DNA (a biological repair mechanism)

Cyclobutane pyrimidine dimer (CPD) can be repaired by: CPD photolyase Enzyme binds to the photodimer and splits it to regenerate the original bases Enzyme requires light to function: photoreactivation

Which repair pathway recognizes DNA damage during transcription? What happens if the damage is not repaired?

DNA damage that stalls transcription is repaired by TC-NER (transcription-coupled nucleotide excision repair). Humans lacking this pathway suffer from Cockayne syndrome. A consequence of this defect is that a cell is much more likely to activate its apoptosis (cell suicide) pathway. Affected individuals are very sensitive to sunlight and have short stature, the appearance of premature aging, and a variety of developmental disorders.

bypass polymerases appear to always be present however

Their access to DNA is regulated so that they are used only when needed The addition of a single ubiquitin (by rad 6) (Ub) monomer to the sliding clamp (PCNA) allows the bypass polymerase to bind to PCNA and begin replicating

In mismatch repair in E. coli, only a mismatch in the newly synthesized strand is corrected. How is E. coli able to recognize the newly synthesized strand? Why does this ability make biological sense?

DNA in E. coli is methylated. To distinguish the old template strand from the newly synthesized strand, the mismatch repair mechanism takes advantage of a delay in the methylation of the new strand. This makes sense as replication errors produce mismatches only on the newly synthesized strand, so the mismatch repair system replaces the "wrong" base on that strand.

Two pathways for nucleotide-excision repair

GG-NER is initiated when a protein complex of XPC and RAD23B recognizes a distorted double helix caused by a damaged base and binds to the opposite strand or TC-NER is initiated when an RNA polymerase complex is stalled by a DNA lesion in the transcribed strand and CSA and CSB bind at this site to form a recognition complex.

synthesis dependent strand annealing (SDSA)

If a double strand break occurs after replication of a chromosomal region in a dividing cell, the damage can be corrected It uses the sister chromatids available in mitosis as the templates to ensure correct repair

Transletion DNA Synthesis

Is a last resort mechanism, which spares the cell the worse fate of an incompletely replicated chromosome Catalyzed by a specialized class of polymerases called bypass polymerases Bypass polymerases are recruited to replication forks that have stalled because of damage in the template strand (a stalled replication fork can initiate a cell death pathway) Bypass polymerases may introduce errors in the course of synthesis that may persist and lead to mutation or that can be corrected by other mechanisms such as mismatch repair

Under what circumstances could nonhomologous end joining be said to be error prone?

NHEJ (nonhomologous end joining) is error prone as some sequence may be lost in the repair process. The consequences of imperfect repair may be far less harmful than leaving the lesion unrepaired. Presumably, this repair pathway evolved because, unless repaired, the broken ends can degrade further, leading to loss of more genetic information. Also, these lesions can initiate potentially harmful chromosomal rearrangements that could lead to cell death.

The molecular consequences of point mutations in a noncoding region

Noncoding regions contain many crucial binding sites At the DNA level: RNA polymerase and its associated factors binds, specific transcription regulators At the RNA level, ribosome binding site, 5' and 3' splice sites for exon joining, sites that regulate translation and localize the mRNA to particular areas and compartments within the cell.

Intercalating agents

Planar molecules that mimic base pairs and are able to slip themselves in (intercalate) between the stacked nitrogen bases can cause insertion or deletion of a single nucleotide pair

transition

Purine is replaced by a purine (A to G or G to A) or Pyrimidine is replaced by a pyrimidine (C to T or T to C)

transversion

Pyrimidine is replaced by a purine or purine is replaced by a pyrimidine

Induced Mutations

Result from the influence of an extraneous factor, either natural or artificial Radiation UV light Natural and synthetic chemical - Production of mutations in the laboratory through exposure to mutagens is called mutagenesis

ionizing radiation

Results in the formation of ionized and excited molecules that can damage DNA E.g. Reactive oxygen species are: superoxide radicals, hydrogen peroxide (H2O2), and hydroxyl radicals (·OH) Can also damage DNA directly rather than through reactive oxygen species. Such radiation may cause breakage of the N-glycosydic bond leading to formation of apurinic or apyrimidinic sites and cause strand breaks Strand breaks are responsible for most of the lethal effects of ionizing radiation

Specific mispairing

Some mutagens do not get incorporated into the DNA but instead alter a base in such a way that it will form a specific mispair. ethylmethanesulfonate adds ethyl groups nitrosoguanidine adds methyl groups

Ames test of aflatoxin B1 mutagenicity

TA100, TA1538 and TA1535 are strains of salmonella bearing different histidine auxotrophic mutations. TA100 strain is highly sensitive to reversion through base-pair substitution. TA1538 and TA1535 strains are sensitive to reversion through frameshift mutations.

Model for the Mismatch repair in E. coli

The first step in mismatch repair is the recognition of the damage in newly replicated DNA by the MutS protein. The binding of this protein to distortions in the DNA double helix caused by mismatched bases initiates the mismatch-repair pathway by attracting three other proteins to the site of the lesion MutH, which performs the crucial function of cutting the strand containing the incorrect base.

Deamination:

The major type of deamination converts cytosine to an altered base, uracil. -Rate is ~ 100 bases per cell per day - Unrepaired U residues will pair with A in replication, resulting on the conversion of a G:C pair into an A:T pair

What are bypass polymerases? How do they differ from the replicative polymerases? How do their special features facilitate their role in DNA repair?

Translesion or bypass polymerases are able to replicate past damaged DNA that otherwise would stall replicative polymerases. They differ from replicative polymerases in that they can tolerate large adducts on the bases (as they have much larger active sites that can accommodate damaged bases), they are much more error-prone (as they lack the 3′ to 5′ proofreading function), and they can only add relatively few nucleotides before falling off. Their main function is to unblock the replication fork, not to synthesize long stretches of DNA that could contain many mismatches.

Translesion synthesis bypasses lesions at stalled replication forks (in E. coli

UV light induces the synthesis of a protein called RecA RecA proteins join the single stranded binding proteins and form a protein-DNA filament (biologically active form of RecA -> signal to induce several genes that encode members of bypass polymerases DNA polymerase III is temporarily replaced by a bypass polymerase (Pol V) that can continue replicating past a lesion

Incorporation of base analogs

When 2-AP is incorporated into DNA by pairing with T, it can generate A.T to G. C transitions by mispairing with C in subsequent replications. Or if 2-AP is incorporated by mispairing with C, then G.C to A.T transitions will result when it pairs with T

Give an example of a DNA-repair defect that leads to cancer.

XP (xeroderma pigmentosum) patients lack nucleotide excision repair and are highly prone to developing pigmented skin cancers. Individuals with HNPCC (hereditary nonpolyposis colorectal cancer) are prone to colorectal cancer due to a loss of the mismatch repair system. Individuals homozygous for mutations in BRCA1 or BRCA2 (breast cancer predisposition genes 1 and 2) are prone to breast cancer due to the loss of repair of double-stranded breaks.

Differentiate between the elements of the following pairs: a. Transitions and transversions b. Synonymous and neutral mutations c. Missense and nonsense mutations d. Frameshift and nonsense mutations

a. A transition mutation is the substitution of a purine for a purine or the substitution of a pyrimidine for a pyrimidine. A transversion mutation is the substitution of a purine for a pyrimidine, or vice versa. b. Both are base-pair substitutions. A synonymous mutation is one that does not alter the amino acid sequence of the protein product from the gene because the new codon codes for the same amino acid as did the nonmutant codon. A neutral mutation results in a different amino acid that is functionally equivalent, and the mutation therefore has no known adaptive significance. c. A missense mutation results in a different amino acid in the protein product of the gene. A nonsense mutation causes premature termination of translation, resulting in a shortened protein. d. Frameshift mutations arise from addition or deletion of one or more bases in other than multiples of three, thus altering the reading frame for translation. Therefore, the amino acid sequence from the site of the mutation to the end of the protein product of the gene will be altered. Frameshift mutations can and often do result in premature stop codons in the new reading frame, leading to shortened protein products. A nonsense mutation causes premature termination of translation in the original reading frame, resulting in a shortened protein.

Which of the following is not possible? a. a nonsynonymous mutation in an intron b. a nonsynonymous mutation in an exon c. an indel mutation in an intron d. an indel mutation in an exon

a. a nonsynonymous mutation in an intron. Introns are removed from the transcripts prior to translation, so they do not code for any amino acids in the protein.

Where in a gene would a 4-bp insertion mutation have the least effect on gene expression? a. introns b. exons c. regulatory regions d. introns and exons

a. introns. The small change in size of an intron would not affect gene expression. Since the intron is removed prior to translation, this insertion would not affect the reading frame of the mRNA.

oncogene mutations

act in the cancer cell as gain of function dominant mutations: Proteins encoded by oncogenes are usually activated in tumor cells (Gain of function) Mutation need be present in only one allele to contribute to tumor formation. The gene in its normal, unmutated form is called protooncogene

nonsense mutation

altered codon signals chain termination

missense (non conservative)

altered codon specifies a chemically dissimilar amino acid

missense mutation (conservative)

altered codon specifies a chemically similar amino acid

synonymous mutation (silent)

altered codon specifies the same amino acid

alkytransferases

are enzymes that directly reverse lesions

Which of the following statements best describe the mismatch repair pathway? a. It is part of the 3′ to 5′ proofreading function of DNA polymerase. b. It acts after DNA replication by recognizing mismatched base pairs. c. It is activated by stalled replication forks. d. It is coupled to transcription.

b. It acts after DNA replication by recognizing mismatched base pairs. The mismatch repair pathway corrects errors that occurred during replication by recognizing mismatched base pairs and repairing them.

Which of the following gene mutations is most likely to have the most severe impact on gene expression? a. a nonsense mutation in the last exon b. a point mutation in an exon c. a point mutation in the splice-donor site of an intron d. a point mutation in the middle of an intron

c. a point mutation in the splice-donor site of an intron. A point mutation in the splice-donor site of an intron would result in the intron sequence inappropriately remaining in the mRNA. This would change the coding sequence of the mRNA, and an incorrect protein product would be made.

Which of the following is/are associated with spontaneous mutation? a. an occurrence of lung cancer due to smoking b. a nonsense mutation in an exon caused by an error in DNA replication c. an indel mutation in an intron caused by replication slippage d. a nonsense mutation in an exon caused by an error in DNA replication, and an indel mutation in an intron caused by replication slippage

d. a nonsense mutation in an exon caused by an error in DNA replication, and an indel mutation in an intron caused by replication slippage. Smoking exposes an individual to mutagens, so it is an example of an induced mutagenesis, not spontaneous mutagenesis. Spontaneous mutations are often the result of errors in replication, either mismatched bases or replication slippage at repeated sequences.

p53 tumor-suppressor gene

encodes a nuclear protein that acts as a transcription factor that represses or stimulates transcription of more than 50 different genes p53 protein is continuously synthesized but rapidly degraded, thus present at low levels Increased levels result from increases in protein phosphorylation, acetylation, and other posttranslational modifications p53 can arrest the cell cycle at several phases Cells lacking p53 are unable to arrest at cell-cycle checkpoints or enter apoptosis in response to DNA damage

cockayne syndrome

have a variety of developmental disorders including dwarfism, deafness, and retardation. age prematurely Patients with Cockayne syndrome have a mutation in one of two proteins called CSA and CSB, which are thought to recognize stalled transcription complexes.

Defend the statement "Cancer is a genetic disease."

he following is a list of observations that argue "cancer is a genetic disease": 1. Certain cancers are inherited as highly penetrant simple Mendelian traits. 2. Most carcinogenic agents are also mutagenic. 3. Various oncogenes have been isolated from tumor viruses. 4. A number of genes that lead to the susceptibility to particular types of cancer have been mapped, isolated, and studied. 5. Dominant oncogenes have been isolated from tumor cells. 6. Certain cancers are highly correlated to specific chromosomal rearrangements. (See Chapter 17 of the textbook.)

NHEJ

in non dividing cells

Aflatoxin B1

is a powerful carcinogen that attaches to guanine The formation of the addition product leads to the breakage of the bond between the base and the sugar Generate an apurinic site

cancer

is an aggregate of cells, all descended from an initial aberrant founder cell (members of a single clone) typically differ from their normal neighbors by a host of phenotypic characters, such as rapid division rate, ability to invade new cellular territories, high metabolic rate, and abnormal shape.

mutation

is an alteration in DNA sequence may be single-base pair substitutions deletion or insertion of one or more base pairs major alteration in chromosomal structure may occur in somatic or germ cells

Xeroderma pigmentosum (XP)

is characterized by the early development of cancers, especially skin cancer and, in some cases, neurological defects. Patients with XP fall into 8 complementation groups, carrying mutations in one of 8 genes encoding proteins XPA through XPG

Sos repair system

is the last resort to minimize DNA damage. DNA synthesis becomes error-prone, inserting random and incorrect nucleotides in places that would normally stall DNA replication SOS repair can itself become mutagenic but allows cells to survive with DNA damage that would otherwise kill itself

Depurination

loss of a purine base) - N-glycosyl linkages to deoxyribose hydrolyze DNA of each human cell loses about 5000 purine bases (A & G) every day

base substitutions

one base pair is replaced by another

Oxidatively damaged bases

oxygen species, such as superoxide radicals, hydrogen peroxide (H2O2), and hydroxyl radicals (·OH), are produced as by-products of normal aerobic metabolism. - These oxygen species can cause oxidative damage to DNA, as well as to precursors of DNA (such as GTP), resulting in mutation

thymine (5-methyluracil)

rather than uracil as the natural pairing partner of adenine in DNA is that spontaneous cytosine deamination events can be recognized as abnormal and then excised and repaired

Double-strand break repair (DSB repair)

reattaches two broken DNA strands 1)Nonhomologous end joining (NHEJ) 2)Homologous recombination repair