Danni al DNA e meccanismi di riparazione

Le mutazioni del DNA

Mutations and Consequences

Mutation refers to the change in a DNA sequence, which may involve only a few bases or

the large-scale chromosome abnormality. This section covers the small-scale mutations

(substitution, deletion, insertion) and the exon skipping that results from mutation at the

splice site.

Substitution

In the substitution mutation, one or more nucleotides are substituted by the same number of

different nucleotides. In most cases, only one nucleotide is changed. Based on the change

in the nucleotide type, the substitution mutation may be divided into transition and

transversion mutations. Based on the consequence of mutation, the substitution mutation

may be grouped into silent, missense and nonsense mutations

Deletion

The deletion mutation involves elimination of one or more nucleotides from a DNA

sequence. It may cause frameshift, producing a non-functional protein.

Note that deletion and insertion mutations often occur in the repetitive sequence. As

explained in the next section, they are usually caused by "replication slippage".

Insertion

In the insertion mutation, one or more nucleotides are inserted into a sequence. If the number

of inserted bases is not a multiple of 3, it will cause frameshift, resulting in serious

consequences. As shown in the following table, non-frameshift insertions may also cause

diseases.

The substitution mutation.

(a) Illustration of transition (blue) and transversion (red) mutations. In the transition mutation,

a pyrimidine (C or T) is substituted by another pyrimidine, or a purine (A or G) is substituted

by another purine. The transversion mutation involves the change from a pyrimidine to a

purine, or vice versa.

(b) Examples of silent, missense and nonsense mutations. The silent mutation does not

produce any change in the amino acid sequence, the missense mutation results in a different

amino acid, and the nonsense mutation generates a stop signal.

Real examples of deletion mutations which cause diseases. (a) Deletion of "T" from the

sequence "TTTTT" in the CFTR gene.

(b) Deletion of "AT" from the sequence "ATAT" in the CFTR gene.

(c) Deletion of "TTG" from the sequence "TTGTTG" in the FIX gene.

(d) Deletion of "ATAG" from the sequence "ATAGATAG" in the APC gene.

Examples of diseases caused by insertion mutation.

Mutation by Replication Errors

Replication errors are the main source of mutations. It has been estimated that uncorrected

replication errors occur with a frequency of 10-9 - 10-11 for each nucleotide added by DNA

polymerases. Since a cell division requires synthesis of 6 X 109 nucleotides, the mutation

rate is about one per cell division.

A commonly observed replication error is the replication slippage, which occurs at the

repetitive sequences when the new strand mispairs with the template strand. The

microsatellite polymorphism is mainly caused by the replication slippage. If the mutation

occurs in a coding region, it could produce abnormal proteins, leading to diseases. The

Huntington's disease is a well known example.

Exon skipping

Example of exon skipping. Splicing of an intron requires an essential signal:

"GT........AG". If the splice acceptor site AG is mutated (e.g., A to C in this figure), the

splicing machinery will look for the next acceptor site. As a result, the exon between two

introns is also removed.

The mutation

caused by

replication

slippage. In this

figure, mispairing

involves only one

repeat. In fact, the

slippage could cause

several repeats to

become

unpaired. (a) Normal

replication. (b)

Backward slippage,

resulting in the

insertion

mutation. (c)

Forward slippage,

resulting in the

deletion mutation.

Mutation Mechanisms

Mutations may be caused by external factors (UV radiation, exposure to chemical agents,

etc.) or spontaneous cellular processes (accidental deamination, replication errors, etc.).

Examples of deamination which involves the removal of an amino group. Accidental

deamination may change the cytosine to uracil, or the methylated cytosine to thymine.

DNA Repair Mechanisms

There are three major DNA repairing mechanisms (damage removal): base excision,

nucleotide excision and mismatch repair.

Proteins involved in the DNA repairing of E. coli.

Tipo Danno Proteine di riparazione

Aggiramento del danno

Sintesi translesione del DNA Dimero di pirimidina o sito

apurinico

DNA pol. IV e V in E. coli

Pol., , , e negli esseri

umani

Inversione del danno

Fotoriattivazione Dimeri di pirimidina DNA fotoliasi

Rimozione di gruppi metilici O6-Metilguanina Metiltransferasi

Rimozione del danno

Riparazione per escissione

delle basi

Base danneggiata DNA glicosilasi

Riparazione delle basi male

appaiate

Errori di replicazione MutS,MutL, e MutH in E. coli

MutS, MutL e EXO1 negli

esseri umani

Riparazione per escissione dei

nucleotidi

Dimeri di pirimidina

Grandi addotti sulle basi

UvrA, UvrB, UvrC e UvrD in

E. coli

XPA, XPB, XPC, XPD,

ERCC1/XPF e XPG negli

esseri umani

Riparazione per rottura a

doppio filamento

Rotture a doppio filamento RecA e REcBCD in E. coli

Complesso MRN, Rad51,

BRCA1, BRCA2, XRCC3,

ecc. negli esseri umani per la

ricombinazione omologa

Proteine Ku, Artemis/DNA-

PKCS, XRCC4 negli esseri

umani per l’unione non

omologa delle estremità

Sistemi di riparazione

del DNA

Cytosine is one of four bases in DNA molecules. As shown in the above figure, it may be

mutated to uracil by deamination. Since uracil is not part of DNA, this mutation can easily

be detected and repaired by base excision. Suppose DNA, like RNA, were made up of

uracil, then the cytosine to uracil mutation could be corrected only by mismatch repair

which is very inefficient. This may explain why DNA chooses thymine, instead of uracil,

even though the chemical structure of uracil is simpler than thymine.

Base excision

DNA's bases may be modified by deamination or alkylation. The position of the

modified (damaged) base is called the "abasic site" or "AP site". In E.coli, the DNA

glycosylase can recognize the AP site and remove its base. Then, the AP endonuclease

removes the AP site and neighboring nucleotides. The gap is filled by DNA polymerase I

and DNA ligase.

Nucleotide excision

In E. coli, proteins UvrA, UvrB, and UvrC are involved in removing the damaged

nucleotides (e.g., the dimer induced by UV light). The gap is then filled by DNA

polymerase I and DNA ligase. In yeast, the proteins similar to Uvr's are named RADxx

("RAD" stands for "radiation"), such as RAD3, RAD10. etc.

Modello della sintesi translesione

del DNA

Riparazione del DNA mediante rimozione dei gruppi metilici

Via di riparazione

per escissione

delle basi nelle

cellule dei

mammiferi

Via di riparazione delle

basi male appaiate

nelle cellule dei mam-

miferi

Via di riparazione per

escissione dei nucleotidi

nei mammiferi

DNA repair by base excision.

DNA repair by nucleotide excision.

Mismatch repair

To repair mismatched

bases, the system has

to know which base is

the correct one. In E.

coli, this is achieved

by a special methylase

called the "Dam

methylase", which can

methylate all adenines

that occur within

(5')GATC

sequences. Immediate

ly after DNA

replication, the

template strand has

been methylated, but

the newly synthesized

strand is not

methylated yet. Thus,

the template strand

and the new strand can

be distinguished.

Mismatch repair. The repairing process begins with the protein MutS which binds to mismatched base

pairs. Then, MutL is recruited to the complex and activates MutH which binds to GATC

sequences. Activation of MutH cleaves the unmethylated strand at the GATC

site. Subsequently, the segment from the cleavage site to the mismatch is removed by

exonuclease (with assistance from helicase II and SSB proteins). If the cleavage occurs on the

3' side of the mismatch, this step is carried out by exonuclease I (which degrades a single

strand only in the 3' to 5' direction). If the cleavage occurs on the 5' side of the mismatch,

exonuclease VII or RecJ is used to degrade the single stranded DNA. The gap is filled by

DNA polymerase III and DNA ligase.

The distance between the GATC site and the mismatch could be as long as 1,000 base

pairs. Therefore, mismatch repair is very expensive and inefficient.

Mismatch repair in eukaryotes may be similar to that in E. coli. Homologs of MutS and MutL

have been identified in yeast, mammals, and other eukaryotes. MSH1 to MSH5 are

homologous to MutS; MLH1, PMS1 and PMS2 are homologous to MutL. Mutations of

MSH2, PMS1 and PMS2 are related to colon cancer.

In eukaryotes, the mechanism to distinguish the template strand from the new strand is still

unclear.

Origin of CpG (CG) islands

The CG island is a short stretch of DNA in which the frequency of the CG sequence is

higher than other regions. It is also called the CpG island, where "p" simply indicates

that "C" and "G" are connected by a phosphodiester bond.

CpG islands are often located around the promoters of housekeeping genes (which

are essential for general cell functions) or other genes frequently expressed in a

cell. At these locations, the CG sequence is not methylated. By contrast, the CG

sequences in inactive genes are usually methylated to suppress their expression.

The methylated cytosine may be converted to thymine by accidental

deamination. Unlike the cytosine to uracil mutation which is efficiently repaired, the

cytosine to thymine mutation can be corrected only by the mismatch repair which is

very inefficient. Hence, over evolutionary time scales, the methylated CG sequence

will be converted to the TG sequence. This explains the deficiency of the CG

sequence in inactive genes.

Inheritance of the DNA

methylation pattern

Any type of cells have

their own methylation

pattern so that a unique set

of proteins may be

expressed to perform

functions specific for this

cell type. Thus, during

cell division, the

methylation pattern should

also pass over to the

daughter cell. This is

achieved by a specific

enzyme called the

maintenance methylase

which can methylate only

the CG sequence paired

with methylated CG.

Mutation by UV light

UV light may cause two

adjacent pyrimidine residues

(cytosine or thymine) to form

a dimer. In a normal cell, the

dimer can be detected by p53,

which then triggers the

repairing process. However,

if p53 itself is mutated and

become non-functional, the

pyrimidine dimer may lead to

mutation.

Figure. Pyrimidine

dimer induced by

UV light. This

figure uses thymine

as an

example. Cytosine

may form a similar

dimer

A possible mechanism for the

mutation induced by UV light.

The UV light first causes two

adjacent cytosine residues to form a

dimer. During DNA replication, both

strands are used as templates to

synthesize new strands. The cytosine

dimer could cause adenine (instead

of the normal guanine) to be

incorporated into the new

strand. Subsequent DNA replication

will produce CC to TT

mutation. Although the cytosine

dimer may eventually be corrected,

the mutation cannot be detected by

the DNA repair system.

Mutation by

Chemical Agents

The chemical agents which may

cause mutation are called

mutagens. Most of them are

also carcinogens.

•Acridines (e.g., proflavin) are positively

charged molecules. They may be inserted

between two DNA strands, thereby altering

DNA's structure and rigidity. As a result,

DNA replication will not be faithful.

•Alkylating agents are chemicals that add an

alkyl group (CnH2n+1) to another

molecule. Alkylation of a base may change

the normal base pairing. For example, the

alkylating agent EMS converts guanine to 7-

ethylguanine which pairs with thymine. The

mispairing will lead to mutation. Some

alkylating agents may also cross-link DNA,

resulting in chromosome breaks.

•Nitrous acid is a deaminating agent that

converts cytosine to uracil, adenine to

hypoxanthine, and guanine to xanthine. The

hydrogen-bonding potential of the modified

base is altered, resulting in mispairing.

•Hydroxylamine and free radicals may

modify base structures, resulting in

mispairing.

Mechanism of mutation induced by 5-bromouracil. This

molecule has two tautomeric isoforms. Its keto form (BUk)

pairs with adenine whereas its enol form (BUe) pairs with

guanine. Suppose in the first replication the keto form was

incorporated into a new DNA strand. During the second

replication, if the keto form undergoes a tautomeric shift to the

enol form, it will cause A:T to G:C mutation.

(a) Base structures induced by free radicals. (b) The base change induced by NH2OH.

Riparazione delle rotture a

doppio filamento

Questi meccanismi operano sia nei procarioti che negli eucarioti.

Il meccanismo di unione non omologa è attivo durante tutto il ciclo cellulare.

L’unione omologa ripara le rotture a doppio filamento a livello delle forcelle di

replicazione.

Unione omologa – ricombinazione omologa

• La ricombinazione omologa svolge ruoli essenziali negli

eucarioti:

• Ricombinazione omologa durante la meiosi.

• Ricombinazione omologa nella trasposizione.

• Ricombinazione omologa per il cambiamento del tipo di

accoppiamento nel lievito.

• Ricombinazione omologa per lo scambio di antigeni nei

tripanosomi.

ATM: ataxia-telangectasia mutated.

Unione omologa –

ricombinazione omologa Proteine coinvolte:

Ser/Thr chinasi nucleare, ATM (trasduttore chiave) aumenta

l’attività di questa chinasi.

ATM fosforila proteine coinvolte nella riparazione del DNA (BRCA1)

e nel controllo del ciclo cellulare (p53; proteina che sopprime i

tumori).

Localizzazione nei siti di rottura di: ATM, proteina Rad52 il

complesso Mre11-Rad50-Nbs1 (MRN) inizia la riparazione (Mre11:

esonucleasi 3’ 5’.

Il DNA a singolo filamento sono riconosciuti da: Rad51.

Altre proteine sono coinvoltenella ricombinazione omologa: Rad54,

Rad55, Rad57, BRCA1 e BRCA2.

Struttura della giunzione di Holliday

(eterodimero)

(nucleasi)

Gli elementi trasponibili del genoma:

trasposoni e retrotrasposoni

Le classi degli elementi trasponibili

Classe Intermedio di trasposizione Esempi

Classe I

Retrotrasposoni LTR RNA Lievito: elementi Ty;

Esseri umani: Retrovirus

endogeni umani (HERV);

Topo: particella A

intracisternali (AP).

Retrotrasposoni non LTR

LINE (autonomi)

SINE (non autonomi)

RNA Esseri umani:

Elementi L1

Elementi Alu

Classe II

Trasposoni di DNA DNA Batteri:

Sequenze di inserzione

Batteriofago Mu

Trasposoni (batterifago Tn7).

Drosophila:

Elementi P.

Mais:

Elementi Ac e Ds.

Invertebrati e vertebrati:

Superfamiglia Tc1/mariner

LINE: elementi nucleari sparsi lunghi.

SINE: elementi nucleari sparsi corti.

LTR: lunghe ripetizioni terminali