INTRACELLULAR SYSTEMS ERRORS producing high-rate mutations

De novo mutations in human genes

in germplasm spontaneous abortions or babies with genetic syndromes or just nothing (Inherited: material for evolution);

In somatic cells spontaneous cell death or improperly functioning cells (never going to be detected) or just nothing (not-inherited: lost in evolution)

or CANCER

Mutations in different places produce different effects

Only a subset of protein variation affects function or charge
Nonsynonymous (aa replacement changes) affect amino acid sequence

Microsatellites 2-5 bp repeats variation in repeat number very diverse

Synonymous variation frequency is several times higher than nonsynonymous no effect on protein

Non-coding variation largely do not affect function similar frequency as to synonymous

www.stats.ox.ac.uk/~mcvean/DTC/Human.ppt

How diverse are humans elsewhere in the genome?

www.stats.ox.ac.uk/~mcvean/DTC/Human.ppt

On average, two genomes differ at about 1 in 1000 bases

This figure is lower in coding regions and higher for regions with short repeated motifs Mutations in genes can affect function detrimentally Slippage during replication and unequal crossing over lead to silent variation in repeat number Humans are one of the least diverse organisms (excepting cheetahs) Species Humans Diversity (percent) 0.1

Chimpanzees
Drosophila simulans E. coli HIV1

1
2 5 30

Human autosomal diversity goes back about 1MY

The mutation rate is about 1.5x10-8 per site per generation Diversity of 0.1% implies an average date of 67,000 generations or about 1.3MY if the average age of reproduction is 20 years (cf chimps) Different stories from different genomes

The MRCA = Most Recent Common Ancestor

The human mtDNA MRCA is estimated to be about 238,000 YA The human Y chromosome MRCA is estimated to be about 91,000 YA (Tang et al. 2002) Why do different parts of the genome tell different stories? Chance Different effective population sizes Different behaviours of men and women Natural selection
www.stats.ox.ac.uk/~mcvean/DTC/Human.ppt

COMMON TYPES OF POINT MUTATIONS

Sources of point mutations in DNA

1) Spontaneous or assisted chemical changes in the DNA

Oxidation (red arrows) Hydrolysis (blue arrows) Methylation (green arrows)

Sources of point mutations in DNA

1) Spontaneous or assisted chemical changes in the DNA
Xantine; Uracil; pairs with T
No deamination Hypoxantine; pairs with C

Deamination as an example

Types of mutations that can occur

Point mutations (single nucleotide changes)
PURINEPURINE or

TRANSITIONS

PYRIMIDINEPIRIMIDINE

MOST COMMON

Pairing is possible due to tautomeric shifts or ionizing that allows mispairing

TRANSVERSIONS purine pyrimidine or pyrimidine purine Pairing is energetically infavourable, but Pur-Pur pairs are possible (G-A)

Other types of mutations that can occur

2. Small insertions/ deletions of 1-5 bp 3. Large chromosomal deletions 4. Translocations Do not forget about them !
By Failure to repair abasic sites

When people talk about carcinogenic mutations,

most often they talk about point mutations.

Point mutations much more tolerable for cell reparation system, and unlikely to awake apoptosis pathway.

Rate of of mutations in human RATES molecular events in genes human cells

Random gain of mutations (low-level; natural cause)
Early stages of natural cancer in elderly; spontaneous mutations in germ plasm

Forced gain of mutations

(median level; X-ray,

chemical carcinogens)
Early stages of cancer and germplasm in exposed people;

Very high rate of mutations (in cells that lost one or more major mechanisms of DNA repair)

Certain genetic syndromes; late stages of almost any cancer

HOW TO CALCULATE RATE OF MUTATIONS IN HUMAN CELLS ?

By HPRT assay

Human HPRT gene

-- Located on chromosome X
-- Encodes the enzyme hypoxanthine phospho ribosyl transferase -- Normal function of HPRT is metabolic salvage of the purines (hypoxanthine and guanine) into nucleotides, inosinic acid, and guanylic acid

Human lymphocytes survive treatment with 6-thioguanine (poison) ONLY if HPRT gene is mutated
6-thioguanine

HPRT ASSAY
The Hypoxanthine Phosphoribsyltransferase Assay. HPRT +/+
6-thioguanine = Poison

So, if cell acquire mutation in HPRT, it become resistant to 6-thioguanine compound

HPRT -/6-thioguanine = OK

We can directly count mutant colonies and compare this number with number of cell seeded on plate

HPRT mutant frequencies

In peripheral T cells In 49 healthy, non-smoking adults rates varied from 0.25 to 9.64 x 10(-6). What made mutation frequencies so different (>10 times)???
CYP1A1, GSTM1 and NAT2 polymorphisms have no influence on HPRT mutation frequencies; mismatch repair genes was also not damaged

Rate of of mutations in human RATES molecular events in genes human cells

Random gain of mutations (low-level; natural cause)
Early stages of natural cancer in elderly; spontaneous mutations in germ cells

Forced gain of mutations

(median level; X-ray,

chemical carcinogens)
Early stages of cancer and germ cells in exposed people;

Very high rate of mutations (cell lost one or more major mechanisms of DNA repair)
Certain genetic syndromes; late stages of almost any cancer

Environmental influences on mutation frequencies?

Yes and no
Many reports with contradictions Maternal alcohol consumption during pregnancy leads to increase of Mut freq. in HPRT assay on T-lymphocytes from newborns
Early pregnancy alcohol RR = 1.84 During pregnancy alcohol RR = 2.99 Smoking during pregnancy have an influence on mutational spectrum, but not on the their frequency
Mutat Res 1999 Dec 17;431(2):279-89

Mutations in HPRT and smoking

Yes, smoking increases mutation frequency
No, smoke does not have any influence

A comparison of mutation frequencies in the K-ras, p53 and HPRT genes between the normal lung tissue of smokers and non-smokers indicates that the rate of mutation in smokers is only ~1.6 fold higher than in non-smokers

HPRT rates among Radiationexposed peoples

Among Hiroshima-Nagasaki survivors (43 Rad in average) 1 mut per 10-8 per base pair per generation

indistinguishable from that of Japanese controls

Chernobyl clean-up workers: 40% increase in mutation rate in the first year after accident,

.then it declined.

So, for lymphocytes classical HPRT assays

do not reveal strong mutational load

Mutagens = (Carcinogens)
Any agent that produces mutations, e.g. tobacco smoke, certain industrial chemicals, ionizing radiation (such as X-rays and ultraviolet rays).

Strong envinronmental carcinogens

ethylene oxide ; 1,3-butadiene ; benzene

JUST marginal increase in HPRT mutability when measured as in vivo exposure (on plant workers populations)
In the same time cell line-based or mice/rat-based assays with direct addition of carcinogen show

strong increase in HPRT mutability

polycyclic aromatic hydrocarbons (PAHs) derivates can bind and damage DNA

In the Liver

Oxidized form of bp

Aflatoxin B1
certain strains of the fungi Aspergillus flavus and A. parasiticus

Prevention trial in China

oltipraz, an inducer of Phase 2

metabolizing enzymes, significantly increased biomarkers of aflatoxin detoxification

Rate of of mutations in human RATES molecular events in genes human cells

Random gain of mutations (low-level; natural cause)
Early stages of natural cancer in elderly; spontaneous mutations in germplasm

Forced gain of mutations

(median level; X-ray,

chemical carcinogens)
Early stages of cancer and germplasm in exposed people;

Very high rate of mutations (cell lost one or more major mechanisms of DNA repair)
Certain genetic syndromes; late stages of almost any cancer

Amount and type of damage can be handled Activation of the survival response network

Damage is excessive and/or irreparable Activation of the apoptosis

Low fidelity repair

Repair

Give up

CELL CANCER CELL DEATH SURVIVAL Nature Reviews Cancer 3; 155-168 (2003); doi:10.1038/nrc1011 ATM AND RELATED PROTEIN KINASES: SAFEGUARDING GENOME INTEGRITY

DNA repair
1/1000 bp of newly synthesized DNA is incorrect but most of mutations are fixed on spot

Only 1/1,000,000 bp are actually miscopied (because of help of DNA repair mechanisms)

Loss of DNA repair mechanisms results in genomic instability, resulting in massive amount of genetic mutations

Many syndromes connected to mutations in reparation-related genes are associated with increase in cancer incidence

Physiological importance of checkpoint pathways

ATM (ataxia telangiectasia) as example

Ataxia:

1 in 40,000-300,000 live births:

loss of motor control owing to Purkinje cell loss, masked faces; oculomotor apraxia

Telangectasias:
Dilated small blood vessels; Skin & Ocular; Onset 4 to 6 years

Immune deficiencies:
Recurrent respiratory infections; T-cell function is reduced

High frequencies of cancer: 14q+ leukemia (T-CLL) B-cell lymphoma

www.neuro.wustl.edu/neuromuscular/ ataxia/dnarep.html

Why ATM defect leads not only to cancer, but also to organismal defects?
cells lacking ATM fail to execute many of the cellular responses to DNA damage. BUT even without any DNA-damaging agent:

1.

during normal cell duplication numerous chromosome defects occur

2.
ATM in oxidative stress

(DNA replication errors, such as double-strand breaks (DSBs))

stalled replication forks 3.

(Cells function in highly reactive chemical environment)

long-lived, terminally differentiated cells such as Purkinje cells are damaged

DSBs in normal VDJ recombination and in meiosis also needs repair.

Concepts of checkpoint
1. Historical DNA damage checkpoints is a NON-ESSENTIAL regulatory pathways that control the ability of cells to arrest the cell cycle in response to DNA damage, allowing time for repair.

2. Modern
Several checkpoint genes (ATR, CHK1) are essential for cell and organism survival

Checkpoint pathways are not only surveyors of occasional damage,

but are firmly integrated components of cellular physiology.

Damage checkpoint pathways

Control cell-cycle arrest
activate DNA repair pathways

Control the movement of DNA repair proteins to sites of DNA damage

Activate different transcriptional programms

control telomere length

induce of cell death by apoptosis

Survival of ATM +/- and ATM-/- cells after IR

HUMAN
Normal lymphocytes ATM +/- mice +/- parents of ATM patient

MICE
Normal mice

ATM -/- mice

ATM patient

Kevin Spring et al., Nature genetics 2002

ATM is a true Tumor Suppressor Gene

ATM itself is 1 in 40,000-300,000 live births (different for different nations) Heterozygotes in populations: 0.35% to 1% Breast cancer susceptible (9-16-time increase in comparison to population) 60% of women with the ATM gene mutation will develop cancer

by the age of

70.

ATM carriers develop breast cancer in response to radiation (due to enchanced radiosensitivity of the cells)
Tuberculosis screening and mammogramms are harmful for ATM carriers

ATM mutations in Ataxia-telangioectasia syndrome

locus.umdnj.edu/nigms/ charmut/atmut.html

ATM acts like any normal kinase

(via phosphorylation)
SUBSTRATES
Chk2, which is involved in control of both the G1/S and G2/M cell-cycle checkpoints

BRCA1 NBS1
tumor suppressor p53
BIN-BING S. ZHOU AND STEPHEN J. ELLEDGE

This processes is similar in their basics non-homologous end-joining machinery (NHEJ)

Homologous recombination V(D)J recombination

V-SEGMENT

Non-homologous end-joining (NHEJ)

DSB

DSB

D-SEGMENT

Rad50, Mre11, Xrs2 complex

Resection Rad52

Ku70, Ku80
RECOMBINATION APPARATUS (RAG1, RAG2)

DNA-PKcs

Strand invasion

GENERATION OF DSBs

Rad50, Mre11, NBS1 complex

Rad51; BRCA2 DNA synthesis

PROCESSING, LIGATION BY NHEJ SYSTEM

RECRUITMENT of NBS1

Ligation, branch migration, Holliday junction resolution

CODING JOIN SIGNAL JOIN

XRCC4/ Ligase IV complex

RECRUITMENT Of XRCC4 and DNA ligase

Ligation

http://www.welc.cam.ac.uk/~spjlab/M7_SPJ_NHEJ/27

Multiple potential roles for Ku/DNA-PKcs in NHEJ

http://www.welc.cam.ac.uk/~spjlab/M7_SPJ_NHEJ/27

MRE11 gene

Chromosome 11q21

Mutated in Ataxia-telangiectasia-like disorder (ATLD) with milder course of disease

Cerebellar degeneration present

Unfortunately, DNA-PKs are not activators of the global DNA damage response (they respond only on DS breaks) It is biochemically difficult to draw the line between sensors and effectors

Multiple potential roles for Ku/DNA-PKcs in NHEJ

http://www.welc.cam.ac.uk/~spjlab/M7_SPJ_NHEJ/27

Nijmegen breakage syndrome (NBS1 -/- defects)

130 cases found worldwide

Growth retardation, intellectual impairment Immune deficiencies

Gonadal failures
NO clinical hallmarks of AT: (no cerebellar ataxia, no telangiectasia, no elevated serum alpha-fetoprotein levels

Cellular defects: spontaneous chromosomal instability, sensitivity to ionizing radiation (IR), and radioresistant DNA synthesis Cancer predisposition (leukaemia, lymphoma, neuroblastoma) 50-fold risk of early-onset common cancers and a greater than 1000-fold risk of lymphoma