Major types of mutations

Major types of mutations

Basis of classification

Mutation typeMajor features

OriginSpontaneous

Induced

Absence of known mutagen

Presence of known mutagen

cell typeSomatic

Germ-line

Non-reproductive cells

Reproductive cells

expressionConditional

Unconditional

Under restrictive conditions

Under permissive conditions

Effect on functionLoss-of-function

Hypomorphic

Hypermorphic

Gain-of-function

Eliminating normal function

Reducing normal function

Increasing normal function

Expressed at incorrect time or in inappropriate cell type

Major types of mutations

Basis of classification

Major types Major features

Molecular changeTransition

Transversion

Insertion

Deletion

Pyrimidine (T,C) to Pyrimidine

Pyrimidine to purine (or opposite)

One or more nucleotides present

One or more missing nucleotides

Effect on translation

Silent

Missence

Nonesense

Frameshift

No change in amino acid

Change in amino acid

Creating stop codon

Shift reading of codon incorrectly

• Example for conditional mutation is temperature-sensitive

Mutation:

Usually the restrictive temperature is high (29°indrosophila)

and the phenotype is mutant above it. The permissive

temperature is low (18° in drosophila) and the phenotype is

wildtype under it.• loss-of-function is also called knock out or null mutation.• A common type of frameshift mutation is a single-base

addition or deletion.

CUG CUG CUG

Leu Leu Leu

CUG CAU GCU G

Leu His Ala

Example (PAH)phenylalanine hydroxylase

CGG = codon on mRNA for Argenine (AA)UGG = codon on mRNA for Tryptophan (AA)

wild type mutantDNA: 5’ ……CGG…… .…..TGG……3 ’

DNA(T): 3’……GCC…… .…..ACC……5’

mRNA: 5’……CGG…… ……UGG......3’So the Aa (Argenine) will be replaced by the Aa

( Tryptophan), that means mutant protein.

Fragile-X syndrome• X-linked form of mental retardation shows

dynamic mutations. The chromosome X tends to fracture near the end of the long arm and it is called fragile-X and the disease is called fragile-X syndrome.

• This syndrome affects 1 in 2500 children and is second only to Down syndrome as a cause of inherited mental impairment.

• Males are more affected than females.

• The molecular basis is trinucleotide repeat of the form CGG (or CCG on the other strand) in the part where the breakage takes place.

• Normal X-chromosome has 6 to 54 repeating unit. Affected persons have 230 to 2300.

• The premutation has an intermediate number of copies ranging from 54 to 230.

• The premutation increases in copy number when transmitted through only females and

is called trinucleotide expansion. This occurs not in germ line but in somatic cells of the early embryo.

• Amplification to 230 copies or more causes silence to the gene FMR1 (fragile-mental retardation-1) which is expressed usually in brain and testes.

• Different extent of amplification in different somatic cells accounts for the variation in severity of this syndrome among affected people.

• The molecular mechanism of trinucleotide expansion is a process called replication slippage (also called slipped-strand mispairing).

• The mechanism of inactivation of FMR1 is Methylation of Cytosine in the full mutation.

• FMR1 protein regulates translation of some mRNA related to development of facial bones and the nervous system. FMRP also functions in learning and memory.

DNA damage can be repaired • Mismach-repair system fixes incorrectly matched

base pairs. • AP endonuclease system repairs nucleotide sites at

which the base has been lost (Apyrimidine site and apurine site).

• Special enzymes repair damage caused to DNA by ultraviolet light.

• Postreplication repair skips over damaged bases.

Cell cycle

The cell cycle is divided into three-part interphase composed of G1 (gap1), S (DNA synthesis), and G2 (gap2), followed by M (mitosis).

Mitosis functions

The essential functions of mitosis are:

1) Replication of DNA once per cycle.

2) Distribution of the replicas (the sister chromatids) equally to the two daughter cells.

Methods to study cell cycle control

• Genetic control has been approached via the methods of biochemistry, cell biology, and genetics.

• Budding yeast, Saccharomyces cerevisiae (S. cerevisiae) is good example for experiments on cell cycle control.

• It has 5538 genes and we can analyze the transcription pattern by gene microarray.

Cell division cycle (cdc) mutants

• Cdc mutant is a mutant whose phenotype is to arrest the cell cycle at a specific point.

• Cdc mutants are wildtype at 23° ( the permissive temperature) and unable to complete cell cycle at 36° ( the restrictive temperature).

• Cdc13 causes arrest at the G2/M boundary because of a defect of telomere processing.

Cdc13 mechanism

• So after the temperature shift, single cells or cells with small buds will give a pair of large cells configuration.

• Cells which were nearing the end of division and had large buds will give quartets after the temperature shift.

Cyclins and cyclin-CDK

• In the early stages of cell cycle, progression from one phase to the next is controlled by characteristic protein complexes that are called cyclin-CDK complexes.

• They are composed of cyclin subunits combined with cyclin-dependent protein kinase (CDK) subunits.

Routes of regulation

1) Phosphorylation:

• When cyclin subunit binds to the protein substrate, the CDK component phosphorylates the substrate.

• Phosphorylation activates some proteins and inactivate others.

• Phosphorylation of different site might inactivate the same activated protein.

2 (Dephosphorylation

Phosphatase enzymes dephosphorylate proteins that CDKs have phosphorylated, reversing the effects of CDKs.

Some important Cyclin-Cdks

Cyclin D-Cdk4 and cyclin D-Cdk6complexes appear in the early or middle part of G1.

They promote entry into S phase .

Cyclin A-Cdk2 and cyclin E-Cdk2 appear later in G1.

Cyclin B-Cdk2 complex carries the cell through the G2/M transition.

Retinoblastoma (RB) protein

The normal role of RB is to maintain cycling cells at a point in G1 called the G1 restriction point or start until the cell has attained proper size.

RB binds to the transcription factor E2F which is needed for further progression in the cell cycle.

RB is phosphorylated by D-Cdk4,6, E-Cdk2 kinase as cells approach G1/S transition.

RB phosphorylation eliminates its ability to bind the E2F transcription factor.

Release of E2F results in transcription of enzymes responsible for DNA synthesis

and E2F itself (as positive autoregulation) .

Through S-phase, E2F is phosphorylated by cyclin A-Cdk2 and inhibits it binding to DNA, so inactivating its function as a transcription factor.

Cyclin B-Cdk2 complex (also called maturation promoting factor) controls the progression from G2 to M transition.

Protein degradation (proteolysis) helpsỊ

Cell cycle go forward by protein degradation that complements the periodic activation of cyclin-CDK complexes.

Progression of cell cycle needs destruction of previous proteins.

Protein degradation entails:

-Sister chromatids to separate.

-Cell to exit from mitosis-.

Checkpoints allow repair or death

A DNA damage checkpoint. (G1/S checkpoint)

A centrosome duplication checkpoint. (G2/M checkpoint)

A spindle checkpoint. (Anaphase checkpoint)

P53 for DNA damage checkpoint

DNA damage checkpoint acts via three stages in the cell cycle:

G1/S transition

S period

G2/M boundary

P53 transcription factor

It is responsible to stress in general, and to DNA damage in particular.

In normal cells, activated p53 protein level is very low. However, p53 mRNA and p53 protein are present.

P53 must go phosphorylation and acetylation to be active.

Mdm2 and p53

The protein Mdm2 inactivates p53 by preventing it from phosphorylation and subsequent steps of its activation.

Activated P53 triggers the transcription of a number of genes and the repression of others.

Genes activated by p53

14-3-3 σ which plays a role to arrest cell at G2/M boundary.

P21 which plays a role at G1/S transition and S checkpoint.

Apaf1 and Bax which promote apoptosis.

Maspin which inhibits angiogenesis (formation of blood vessels) and metastasis.

Apoptosis

Means programmed cell death. (suicide)

DNA damage also triggers activation of the pathway of apoptosis.

A cascade of proteins involved in the lysis of the cell initiate suicide.

They are called caspases.

Oncogenes

Oncogene is a gain-of-function mutation in a cellular gene, called proto-oncogene, whose normal function is to promote proliferation or inhibit apoptosis; oncogenes are often associated with tumor progression.

Oncogenes and Bcl2

Oncogenes can increase the level of activated (phosphorylated) Bcl2, which prevents apoptosis and allows the affected

cells to grow and divide indefinitely .

Tumor suppressor genes

Tumor suppressor gene is a loss-of-function mutation in a cellular gene, whose normal function is to inhibit cell division or to

activate apoptosis .

Tumor suppressor genes

Tumor suppressor gene

alterationconsequence

p53mutationLoss of G1/S, S, and G2/M checkpoint

p21mutationLoss of G1/S and S checkpoint

RBmutationPromote proliferation; E2F uninhibited

BaxmutationFailure of apoptosis

Centrosome duplication checkpoint and the spindle checkpoint

Centrosome is the organelle around which the bipolar spindle is organized.

Failure in centrosome duplication checkpoint might result in polyploidy .

Failure in spindle checkpoint might cause aneuploidy .

APC

APC is the anaphase-promoting complex needed for entry into anaphase.

Correct attachment to the spindle causes inactive spindle checkpoint (inactive Bub, Mad, and Mps) and so, active APC.

Incorrect or unbalanced attachment to the spindle activates spindle checkpoint and

so, inactive APC .

Loss of heterozygosity

Loss of the presence of the wildtype allele, or loss of its function, in a heterozygous cell, enabling the phenotype of a recessive mutant allele to be expressed.