SC435 Genetics Seminar

• Welcome to our Unit 8 Seminar

• We will continue our discussion of mutation and cancer

• The seminar will begin at 9:00PM ET

Unit 8

• Discussion board• Unit 8 Quiz• Unit 8 Exam

• Looking ahead– Final Project

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Unit Readings

Chapter 12

Chapter 13

Mutations• A mutation is any heritable change in the genetic material

• Mutations are classified in a variety of ways

• Most mutations are spontaneous: they are random, unpredictable events

• Each gene has a characteristic rate of spontaneous mutation, measured as the probability of a change in DNA sequence in the time span of a single generation

Table 12.1

Mutations• Rates of mutation can be increased by treatment with a chemical

mutagen or radiation, in which case the mutations are said to be induced

• Mutations in cells that form gametes are germ-line mutations; all others are somatic mutations

• Germ-line mutations are inherited; somatic mutations are not

• A somatic mutation yields an organism that is genotypically a mixture (mosaic) of normal and mutant tissue

Mutations• Among the mutations that are most useful for genetic

analysis are those whose effects can be turned on or off by the researcher

• These are conditional mutations: they produce phenotypic changes under specific (permissive conditions) conditions but not others (restrictive conditions)

– Temperature-sensitive mutations: conditional mutation whose

expression depends on temperature

Mutations• Mutations can also be classified according to their effects on gene

function:

A loss-of-function mutation (a knockout or null) results in complete gene inactivation or in a completely nonfunctional gene product

A hypomorphic mutation reduces the level of expression of a gene or activity of a product

A hypermorphic mutation produces a greater-than-normal level of gene expression because it changes the regulation of the gene so that the gene product is overproduced

A gain-of-function mutation qualitatively alters the action of a gene. For example, a gain-of-function mutation may cause a gene to become active in a type of cell or tissue in which the gene is not normally active.

Mutations• Mutations result from changes in DNA

• A base substitution replaces one nucleotide pair with another

• Transition mutations replace one pyrimidine base with the other or one purine base with the other. There are four possible transition mutations

Mutations• Transversion mutations replace a pyrimidine with a

purine or the other way around. There are eight possible transversion mutations

• Spontaneous base substitutions are biased in favor of transitions:

• Among spontaneous base substitutions, the ratio of transitions to transversions is approximately 2:1

Fig. 12.19

Mutations• Mutations in protein-coding regions can change an amino acid,

truncate the protein, or shift the reading frame:

• Missense or nonsynonymous substitutions result in one amino acid being replaced with another

• Synonymous or silent substitutions in DNA do not change the amino acid sequence

• Silent mutations are possible because the genetic code is redundant

Mutations• A nonsense mutation creates a new stop codon

• Frameshift mutations shift the reading frame of the codons in the mRNA

• Any addition or deletion that is not a multiple of three

nucleotides will produce a frameshift

Sickle-cell anemia The molecular basis of sickle-cell anemia is a mutant gene for -

globin

The sickle-cell mutation changes the sixth codon in the coding sequence from the normal GAG, which codes for glutamic acid, into the codon GUG, which codes for valine

Sickle-cell anemia is a severe genetic disease that often results in premature death

The disease is very common in regions where malaria is widespread because it confers resistance to malaria

Spontaneous Mutations• Mutations are statistically random events—there is no way of

predicting when, or in which cell, a mutation will take place

• The mutational process is also random in the sense that whether a particular mutation happens is unrelated to any adaptive advantage it may confer on the organism in its environment

• A potentially favorable mutation does not arise because the organism has a need for it

Spontaneous Mutations• Several types of experiments showed that adaptive

mutations take place spontaneously and were present at low frequency in the population even before it was exposed to the selective agent

• One experiment utilized a technique developed by Joshua and Esther Lederberg called replica plating

• Selective techniques merely select mutants that preexist in a population

Fig. 12.13

Mutation Hot Spots

• Mutations are nonrandom with respect to position in a gene or genome

• Certain DNA sequences are called mutational hotspots because they are more likely to undergo mutation than others

• For instance, sites of cytosine methylation are usually highly mutable

Mutagenes• Almost any kind of mutation that can be induced by a mutagen can also occur

spontaneously, but mutagens bias the types of mutations that occur according to the type of damage to the DNA that they produce

DNA Repair Mechanisms• Many types of DNA damage can be repaired• Mismatch repair fixes incorrectly matched base pairs• The AP endonuclease system repairs nucleotide sites at which

the base has been lost• Special enzymes repair damage caused to DNA by ultraviolet light• Excision repair works on a wide variety of damaged DNA• Postreplication repair skips over damaged bases

Mismatch Repair• Mismatch repair fixes incorrectly matched base pairs: a segment

of DNA that contains a base mismatch excised and repair synthesis followed

• The mismatch-repair system recognizes the degree of methylation of a strand and preferentially excises nucleotides from the undermethylated strand

• This helps ensure that incorrect nucleotides incorporated into the daughter strand in replication will be removed and repaired.

Mismatch Repair

• The daughter strand is always the undermethylated strand because its methylation lags somewhat behind the moving replication fork

Fig. 12.27

Mismatch Repair

• The most important role of mismatch repair is as a “last chance” error-correcting mechanism in replication

Fig. 12.26

AP Repair• Deamination of cytosine creates uracil which is

removed by DNA uracil glycosylase from deoxyribose sugar. The result is a site in the DNA that lacks a pyrimidine base (an apyrimidinic site)

• Purines in DNA are somewhat prone to hydrolysis, which leave a site that is lacking a purine base (an apurinic site)

• Both apyrimidinic and apurinic sites are repaired by a system that depends on an enzyme called AP endonuclease

Fig. 12.28

Excision Repair• Excision repair is a ubiquitous,

multistep enzymatic process by which a stretch of a damaged DNA strand is removed from a duplex molecule and replaced by resynthesis using the undamaged strand as a template

Fig. 12.29

Postreplication repair

• Sometimes DNA damage persists rather than being reversed or removed, but its harmful effects may be minimized. This often requires replication across damaged areas, so the process is called postreplication repair

Fig. 12.30

Ames test• In view of the increased number of chemicals used and

present as environmental contaminants, tests for the mutagenicity of these substances has become important

• Furthermore, most agents that cause cancer (carcinogens) are also mutagens, and so mutagenicity provides an initial screening for potential hazardous agents

• A genetic test for mutations in bacteria that is widely used for the detection of chemical mutagens is the Ames test

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• There are two major parts in the cell cycle:

Interphase: G1 = gap1 S = DNA synthesis G2 = gap2

Mitosis: M

• There are two essential functions of the cell cycle: To ensure that each chromosomal DNA molecule is

replicated only once per cycle To ensure that the identical replicas of each chromosome

are distributed equally to the two daughter cells

The Cell Cycle

30Fig. 13.1

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The Cell Cycle• The cell cycle is under genetic control

• A fundamental feature of the cell cycle is that it is a true cycle: it is not reversible

• Many genes are transcribed during the cell cycle just before their products are needed

• Mutations affecting the cell cycle have helped to identified the key regulatory pathways

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The Cell Cycle• Progression from one phase to the next is propelled by

characteristic protein complexes, which are composed of Cyclins and cyclin-dependent protein kinases (CDK)

• Expression of mitotic cyclins E, A, and B are periodic, whereas cyclin D is expressed throughout the cell cycle in response to mitosis stimulating drugs (mitogens)

• The cyclin-CDK complexes phosphorylate targeted proteins, changing dramatically their activity

33Fig. 13.8

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The Retinoblastoma Protein• The retinoblastoma (RB) protein controls the initiation of DNA

synthesis.• RB maintains cells at a point in G1 called the G1 restriction point or

start by binding to the transcription factor E2F, until the cell has attained proper size

• If the cycling cell is growing properly and becomes committed to DNA synthesis, several cyclin-CDK complexes inactivate RB by phosphorylation

• After cell enters S phase E2F becomes phosphorylated as well and loses its ability to bind DNA

35Fig. 13.10

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The Cell Cycle• The progression from G2 to M is controlled by a cyclin B-CDK2

complex know as maturation-promoting complex• Protein degradation (proteolysis) also helps regulate the cell cycle.

The anaphase-promoting complex (APC/C), which is a ubiquitin–protein ligase responsible for adding the 76-amino-acid protein ubiquitin to its target proteins and marking them for destruction in the proteasome

• Proteolysis eliminates proteins used in the preceding phase as well as proteins that would inhibit progression into the next

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Checkpoints• Cells monitor their external environments and internal state and

functions

• Checkpoints in the cell cycle serve to maintain the correct order of steps as the cycle progresses; they do this by causing the cell cycle to pause while defects are corrected or repaired

• Checkpoints in the cell cycle allow damaged cells to repair themselves or to self-destruct

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Three Main Checkpoints

• A DNA damage checkpoint

• A centrosome duplication checkpoint

• A spindle checkpoint

39Fig. 13.12

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A DNA Damage Checkpoint• A DNA damage checkpoint arrests the cell cycle when DNA is

damaged or replication is not completed.

• In animal cells, a DNA damage checkpoint acts at three stages in the cell cycle: at the G1/S transition, in the S period and at the G2/M boundary

• The p53 transcription factor is a key player in the DNA damage

checkpoint.

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A DNA Damage Checkpoint• In normal cells, level of activated p53 is very low

• Protein Mdm2 keeps p53 inactivated by preventing phosphorylation and acetylation of p53 and by exporting p53 from the nucleus

• Damaged DNA leads to activation of p53 and its release from Mdm2

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A DNA Damage Checkpoint• Activated p53 triggers transcription of a number of

genes - p21, 14-3-3s, Bax, Apaf1, Maspin,GADD45• DNA damage detected in G1 blocks cell G1/S transition• DNA damage in S phase reduces processivity of DNA

polymerase and gives the cell time for repairProcessivity: number of consecutive nucleotides that replicate before polymerase detaches from template

• DNA damage detected in S or G1 arrests cells at G2/M transition

43Fig. 13.15

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A DNA Damage Checkpoint• DNA damage also triggers activation of a pathway for

apoptosis = programmed cell death

• When the apoptotic pathway is activated, a cascade of proteolysis is initiated that culminates in cell suicide

• The proteases involved are called caspases

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Centrosome Duplication Checkpoint• Monitors spindle formation

• Functions to maintain the normal complement of chromosomes

• Sometimes coordinates with the spindle checkpoint and the exit from mitosis

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The Spindle Checkpoint• Monitors assembly of the spindle and its attachment to

kinetochores

• The kinetochore is the spindle-fiber attachment site on the chromosome

• Incorrect or unbalanced attachment to the spindle activates spindle checkpoint proteins, triggers a block in the separation of the sister chromatids by preventing activation of the anaphase-promoting complex (APC/C)

47Fig. 13.18

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Cancer• Cancer cells have a small number of mutations that

prevent normal checkpoint function

• Cancer is not one disease but rather many diseases with similar cellular attributes

• All cancer cells show uncontrolled growth as a result of mutations in a relatively small number of genes

• Cancer is a disease of somatic cells

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• 1% of cancer cases are familial: show evidence for segregation of a gene in pedigree

• 99% are sporadic: the result of genetic changes in somatic cells

• Within an organism, tumor cells are clonal, which means that they are descendants from a single ancestral cell that became

cancerous.

Cancer

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Cancer Cells vs. Normal Cells• In normal cells, cell-to-cell contact inhibits further

growth and division, a process called contact inhibition

• Cancer cells have lost contact inhibition: they

continue to grow and divide, and they even pile on top of one another

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Cancer Cells vs. Normal Cells • Even in the absence of damage, normal cells cease to

divide in culture after about 50 doublings = cell senescence

• Senescence of normal cells is associated with a loss of telomerase activity: the telomeres are no longer elongated, which contributes to the onset of senescence and cell death

• Cancer cells have high levels of telomerase, which help to protect them from senescence, making them immortal

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Key Mutational Targets• Many cancers are the result of alterations in cell cycle control,

particularly in control of the G1-to-S transition

• These alterations also affect apoptosis through their interactions with p53

• The major mutational targets for the multistep cancer progression are of two types:

Proto-oncogenes

Tumor-suppressor genes

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Key Mutational Targets• The normal function of proto-oncogenes is to promote cell

division or to prevent apoptosis

• The normal function of tumor-suppressor genes is to prevent cell

division or to promote apoptosis

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Familial Cancers• Mutations that predispose to cancer can be inherited

through the germ line

• The presence of this mutation predisposes the individual to cancer, because it reduces the number of additional somatic mutations necessary for a precancerous cell to progress to malignancy

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Li–Fraumeni Syndrome• The Li–Fraumeni syndrome shows clear autosomal dominant

inheritance. However, the affected individuals have a range of different tumors and often have more than one, including osteosarcoma, leukemia, breast cancer, lung cancer, soft-tissue sarcoma, and brain tumors

• A large fraction of Li–Fraumeni families show segregation for a

mutation in the p53 gene.

• A situation analogous to the human Li–Fraumeni syndrome has been created in mice by experimental knockout (loss of function) of the p53gene via the germ-line transformation

56Fig. 13.24