Danni al DNA e meccanismi di riparazione Le mutazioni del DNA

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1 Danni al DNA e meccanismi di riparazione Le mutazioni del DNA

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9 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.

10 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.

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12 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.

13 Examples of diseases caused by insertion mutation.

14 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 for each nucleotide added by DNA polymerases. Since a cell division requires synthesis of 6 X 10 9 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.

15 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.

16 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.

17 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.

18 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.

19 Sistemi di riparazione del DNA Tipo Danno Proteine di riparazione Aggiramento del danno Sintesi translesione del DNA Inversione del danno Dimero di pirimidina o sito apurinico DNA pol. IV e V in E. coli Pol.,,, e negli esseri umani Fotoriattivazione Dimeri di pirimidina DNA fotoliasi Rimozione di gruppi metilici O 6 -Metilguanina Metiltransferasi Rimozione del danno Riparazione per escissione delle basi Riparazione delle basi male appaiate Riparazione per escissione dei nucleotidi Riparazione per rottura a doppio filamento Base danneggiata Errori di replicazione Dimeri di pirimidina Grandi addotti sulle basi Rotture a doppio filamento DNA glicosilasi MutS,MutL, e MutH in E. coli MutS, MutL e EXO1 negli esseri umani UvrA, UvrB, UvrC e UvrD in E. coli XPA, XPB, XPC, XPD, ERCC1/XPF e XPG negli esseri umani 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

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21 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.

22 Modello della sintesi translesione del DNA

23 Riparazione del DNA mediante rimozione dei gruppi metilici

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25 Via di riparazione per escissione delle basi nelle cellule dei mammiferi

26 Via di riparazione delle basi male appaiate nelle cellule dei mammiferi

27 Via di riparazione per escissione dei nucleotidi nei mammiferi

28 DNA repair by base excision.

29 DNA repair by nucleotide excision.

30 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.

31 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.

32 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.

33 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.

34 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

35 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.

36 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 (C n H 2n+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.

37 Mechanism of mutation induced by 5-bromouracil. This molecule has two tautomeric isoforms. Its keto form (BU k ) pairs with adenine whereas its enol form (BU e ) 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.

38 (a) Base structures induced by free radicals. (b) The base change induced by NH 2 OH.

39 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.

40 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.

41 ATM: ataxia-telangectasia mutated.

42 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.

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46 Struttura della giunzione di Holliday

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48 (eterodimero) (nucleasi)

49 Gli elementi trasponibili del genoma: trasposoni e retrotrasposoni

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51 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) Classe II RNA Esseri umani: Elementi L1 Elementi Alu 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

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