genetica per scienze naturali a.a. 05-06 prof s. presciuttini biological repair mechanisms there are...

Genetica per Scienze Naturalia.a. 05-06 prof S. Presciuttini

Biological repair mechanisms There are many potential threats to the fidelity of DNA replication. There are many potential threats to the fidelity of DNA replication.

Not only is there an inherent error rate for the replication of DNA, but Not only is there an inherent error rate for the replication of DNA, but there are also spontaneous lesions that can provoke additional errors. there are also spontaneous lesions that can provoke additional errors. Moreover, mutagens in the environment can damage DNA and greatly Moreover, mutagens in the environment can damage DNA and greatly increase the mutation rate.increase the mutation rate.

Living cells have evolved a series of enzymatic systems that repair Living cells have evolved a series of enzymatic systems that repair DNA damage in a variety of ways. Failure of these systems can lead DNA damage in a variety of ways. Failure of these systems can lead to a higher mutation rate. A number of human diseases including to a higher mutation rate. A number of human diseases including certain types of cancer can be attributed to defects in DNA repair, as certain types of cancer can be attributed to defects in DNA repair, as we shall see later. Let's first examine some of the characterized repair we shall see later. Let's first examine some of the characterized repair pathways and then consider how the cell integrates these systems into pathways and then consider how the cell integrates these systems into an overall strategy for repair.an overall strategy for repair.

We can divide repair pathways into several categories.We can divide repair pathways into several categories.


Prevention of errors before they happen Some enzymatic systems neutralize potentially damaging compounds Some enzymatic systems neutralize potentially damaging compounds

before they even react with DNA. One example of such a system is before they even react with DNA. One example of such a system is the detoxification of superoxide radicals produced during oxidative the detoxification of superoxide radicals produced during oxidative damage to DNA: the enzyme damage to DNA: the enzyme superoxide dismutasesuperoxide dismutase catalyzes the catalyzes the conversion of the superoxide radicals into hydrogen peroxide, and the conversion of the superoxide radicals into hydrogen peroxide, and the enzyme enzyme catalasecatalase,, in turn, converts the hydrogen peroxide into water. in turn, converts the hydrogen peroxide into water. Another error-prevention pathway depends on the protein product of Another error-prevention pathway depends on the protein product of the the mutTmutT gene: this enzyme prevents the incorporation of 8-oxodG, gene: this enzyme prevents the incorporation of 8-oxodG, which arises by oxidation of dGTP, into DNA by hydrolyzing the which arises by oxidation of dGTP, into DNA by hydrolyzing the triphosphate of 8-oxodG back to the monophosphatetriphosphate of 8-oxodG back to the monophosphate

DNA damage products formed after attack by DNA damage products formed after attack by oxygen radicals. dR = deoxyriboseoxygen radicals. dR = deoxyribose


Direct reversal of damage The most straightforward way to repair a lesion, once it occurs, is to reverse it The most straightforward way to repair a lesion, once it occurs, is to reverse it

directly, thereby regenerating the normal base. Reversal is not always possible, directly, thereby regenerating the normal base. Reversal is not always possible, because some types of damage are essentially irreversible. In a few cases, however, because some types of damage are essentially irreversible. In a few cases, however, lesions can be repaired in this way. One case is a mutagenic photodimer caused by lesions can be repaired in this way. One case is a mutagenic photodimer caused by UV light. The cyclobutane pyrimidine photodimer can be repaired by a UV light. The cyclobutane pyrimidine photodimer can be repaired by a photolyasephotolyase that has been found in bacteria and lower eukaryotes but not in humans. The that has been found in bacteria and lower eukaryotes but not in humans. The enzyme binds to the photodimer and splits it, in the presence of certain wavelengths enzyme binds to the photodimer and splits it, in the presence of certain wavelengths of visible light, to generate the original bases. This enzyme cannot operate in the of visible light, to generate the original bases. This enzyme cannot operate in the dark, so other repair pathways are required to remove UV damage. A photolyase dark, so other repair pathways are required to remove UV damage. A photolyase that reverses the 6-4 photoproducts has been detected in plants and that reverses the 6-4 photoproducts has been detected in plants and Drosophila.Drosophila.

Repair of a UV-induced pyrimidine photodimer by a Repair of a UV-induced pyrimidine photodimer by a photoreactivating enzyme, or photolyase. The enzyme photoreactivating enzyme, or photolyase. The enzyme recognizes the photodimer (here, a thymine dimer) and recognizes the photodimer (here, a thymine dimer) and binds to it. When light is present, the photolyase uses binds to it. When light is present, the photolyase uses its energy to split the dimer into the original its energy to split the dimer into the original

monomers.monomers.


Removal of alkyl groups AlkyltransferasesAlkyltransferases also are enzymes taking part in the direct reversal of lesions. also are enzymes taking part in the direct reversal of lesions.

They remove certain alkyl groups that have been added to the O-6 positions of They remove certain alkyl groups that have been added to the O-6 positions of guanine by such agents as NG and EMS. The methyltransferase from guanine by such agents as NG and EMS. The methyltransferase from E. coliE. coli has has been well studied. This enzyme transfers the methyl group from been well studied. This enzyme transfers the methyl group from OO-6-methylguanine -6-methylguanine to a cysteine residue on the protein. When this happens, the enzyme is inactivated, to a cysteine residue on the protein. When this happens, the enzyme is inactivated, so this repair system can be saturated if the level of alkylation is high enough. so this repair system can be saturated if the level of alkylation is high enough.

Alkylation-induced specific mispairing. The Alkylation-induced specific mispairing. The alkylation (in this case, EMS-generated ethylation) alkylation (in this case, EMS-generated ethylation)

of the O-6 position of guanine andof the O-6 position of guanine and the O-4 the O-4 position of thymine can lead to direct mispairing position of thymine can lead to direct mispairing with thymine and guanine, respectively, as shown with thymine and guanine, respectively, as shown here. In bacteria, where mutations have been here. In bacteria, where mutations have been analyzed in great detail, the principal mutations analyzed in great detail, the principal mutations detected are GC → AT transitions, indicating that detected are GC → AT transitions, indicating that the O-6 alkylation of guanine is most relevant to the O-6 alkylation of guanine is most relevant to mutagenesis. mutagenesis.


Excision-repair pathways

Also termed Also termed nucleotide excision nucleotide excision repair,repair, this system includes the this system includes the breaking of a phosphodiester breaking of a phosphodiester bond on either side of the lesion, bond on either side of the lesion, on the same strand, resulting in on the same strand, resulting in the excision of an the excision of an oligonucleotide. This excision oligonucleotide. This excision leaves a gap that is filled by leaves a gap that is filled by repair synthesis, and a ligase repair synthesis, and a ligase seals the breaks. In prokaryotes, seals the breaks. In prokaryotes, 12 or 13 nucleotides are 12 or 13 nucleotides are removed; whereas, in removed; whereas, in eukaryotes, from 27 to 29 eukaryotes, from 27 to 29 nucleotides are eliminated.nucleotides are eliminated.


The excinuclease In In E. coli,E. coli, the products of the the products of the uvrA, B,uvrA, B, and and CC genes constitute the genes constitute the

excinuclease. The UvrA protein, which recognizes the damaged DNA, excinuclease. The UvrA protein, which recognizes the damaged DNA, forms a complex with UvrB and leads the UvrB subunit to the damage forms a complex with UvrB and leads the UvrB subunit to the damage site before dissociating. The UvrC protein then binds to UvrB. Each site before dissociating. The UvrC protein then binds to UvrB. Each of these subunits makes an incision. The short DNA 12-mer is of these subunits makes an incision. The short DNA 12-mer is unwound and released by another protein, helicase II.unwound and released by another protein, helicase II.

The human excinuclease is considerably The human excinuclease is considerably more complex than its bacterial counterpart more complex than its bacterial counterpart and includes at least 17 proteins. However, and includes at least 17 proteins. However, the basic steps are the same as those in the basic steps are the same as those in E. E. colicoli

Schematic representation of events following incision Schematic representation of events following incision by UvrABC exinuclease in by UvrABC exinuclease in E. coli.E. coli.


Excision repair defects in humans Several human genetic diseases are known to be due to repair defects.Several human genetic diseases are known to be due to repair defects. Xeroderma pigmentosum (XP)Xeroderma pigmentosum (XP) results from a defect in any of the results from a defect in any of the

genes (complementation groups) effecting nucleotide excision repair. genes (complementation groups) effecting nucleotide excision repair. People suffering from this disorder are extremely prone to UVinduced People suffering from this disorder are extremely prone to UVinduced skin cancers as a result of exposure to sunlight and have frequent skin cancers as a result of exposure to sunlight and have frequent neurological abnormalities.neurological abnormalities.

Nucleotide excision repair is coupled to transcription. This model for coupled repair in mammalian cells shows RNA polymerase (pink) pausing when encountering a lesion. It undergoes a conformational change, allowing the DNA strands at the lesion site to reanneal.

Protein factors aid in coupling by bringing TFIIH and other factors to the site to carry out the incision, excision, and repair reactions. Then transcription can continue normally.


Specific excision pathways Certain lesions are too subtle to cause a distortion Certain lesions are too subtle to cause a distortion

large enough to be recognized by the large enough to be recognized by the uvrABCuvrABC--encoded general excision-repair system and its encoded general excision-repair system and its counterparts in higher cells. Thus, additional counterparts in higher cells. Thus, additional excision pathways are necessary.excision pathways are necessary.

DNA glycosylase repair pathway (base-excision DNA glycosylase repair pathway (base-excision repair).repair). DNA glycosylasesDNA glycosylases do not cleave do not cleave phosphodiester bonds, but instead cleave N-phosphodiester bonds, but instead cleave N-glycosidic (base–sugar) bonds, liberating the glycosidic (base–sugar) bonds, liberating the altered base and generating an apurinic or an altered base and generating an apurinic or an apyrimidinic site, both called apyrimidinic site, both called AP sites,AP sites, because because they are biochemically equivalent. The resulting they are biochemically equivalent. The resulting Ap site is then repaired by an AP endonuclease Ap site is then repaired by an AP endonuclease repair pathwayrepair pathway


Mismatch repair Some repair pathways are capable of recognizing errors even after Some repair pathways are capable of recognizing errors even after

DNA replication has already occurred. One such system, termed the DNA replication has already occurred. One such system, termed the mismatch repair system,mismatch repair system, can detect mismatches that occur in DNA can detect mismatches that occur in DNA replication. Suppose you were to design an enzyme system that could replication. Suppose you were to design an enzyme system that could repair replication errors. What would this system have to be able to repair replication errors. What would this system have to be able to do? At least three things:do? At least three things:

1. Recognize mismatched base pairs. 1. Recognize mismatched base pairs. 2. Determine which base in the mismatch is the incorrect one. 2. Determine which base in the mismatch is the incorrect one. 3. Excise the incorrect base and carry out repair synthesis. 3. Excise the incorrect base and carry out repair synthesis.

The second point is the crucial property of such a system. Unless it is The second point is the crucial property of such a system. Unless it is capable of discriminating between the correct and the incorrect bases, capable of discriminating between the correct and the incorrect bases, the mismatch repair system could not determine which base to excise. the mismatch repair system could not determine which base to excise. If, for example, a G–T mismatch occurs as a replication error, how If, for example, a G–T mismatch occurs as a replication error, how can the system determine whether G or T is incorrect? Both are can the system determine whether G or T is incorrect? Both are normal bases in DNA. But replication errors produce mismatches on normal bases in DNA. But replication errors produce mismatches on the newly synthesized strand, so it is the base on this strand that must the newly synthesized strand, so it is the base on this strand that must be recognized and excised.be recognized and excised.


DNA methylation To distinguish the old, template strand from the newly synthesized To distinguish the old, template strand from the newly synthesized

strand, the mismatch repair system in bacteria takes advantage of the strand, the mismatch repair system in bacteria takes advantage of the normal delay in the postreplication methylation of the sequence normal delay in the postreplication methylation of the sequence

The methylating enzyme is adenine methylase, which creates 6-The methylating enzyme is adenine methylase, which creates 6-methyladenine on each strand. However, it takes the adenine methyladenine on each strand. However, it takes the adenine methylase several minutes to recognize and modify the newly methylase several minutes to recognize and modify the newly synthesized GATC stretches. During that interval, the mismatch repair synthesized GATC stretches. During that interval, the mismatch repair system can operate because it can now distinguish the old strand from system can operate because it can now distinguish the old strand from the new one by the methylation pattern. Methylating the 6-position of the new one by the methylation pattern. Methylating the 6-position of adenine does not affect base pairing, and it provides a convenient tag adenine does not affect base pairing, and it provides a convenient tag that can be detected by other enzyme systems.that can be detected by other enzyme systems.


Mismatch repairModel for mismatch repair in Model for mismatch repair in E. coli.E. coli. Because DNA is methylated by Because DNA is methylated by enzymatic reactions that recognize the A enzymatic reactions that recognize the A in a GATC sequence, the newly in a GATC sequence, the newly synthesized strand will not be methylated synthesized strand will not be methylated directly after DNA replication.directly after DNA replication.The The hemimethylatedhemimethylated DNA duplex serves DNA duplex serves as a recognition point for the mismatch as a recognition point for the mismatch repair system in discerning the old from repair system in discerning the old from the new strand. Here a G–T mismatch is the new strand. Here a G–T mismatch is shown. The mismatch repair system can shown. The mismatch repair system can recognize and bind to this mismatch, recognize and bind to this mismatch, determine the correct (old) strand determine the correct (old) strand because it is the methylated strand of a because it is the methylated strand of a hemimethylated duplex, and then excise hemimethylated duplex, and then excise the mismatched base from the new the mismatched base from the new strand. Repair synthesis restores the strand. Repair synthesis restores the normal base pair. normal base pair.


The complex MutS-MutH Steps in Steps in E. coliE. coli mismatch repair. mismatch repair.

(1) MutS binds to mispair.(1) MutS binds to mispair. (2) MutH and MutL are recruited to form a (2) MutH and MutL are recruited to form a

complex. MutH cuts the newly synthesized complex. MutH cuts the newly synthesized (unmethylated) strand, and exonuclease (unmethylated) strand, and exonuclease degradation goes past the point of the mismatch, degradation goes past the point of the mismatch, leaving a patch.leaving a patch.

(3) Single-strand-binding protein (Ssb) protects (3) Single-strand-binding protein (Ssb) protects the single-stranded region across from the the single-stranded region across from the missing patch.missing patch.

(4) Repair synthesis and ligation fill in the gap. (4) Repair synthesis and ligation fill in the gap.


Mismatch repair defects in humansHereditary nonpolyposis colorectal cancer (HNPCC)Hereditary nonpolyposis colorectal cancer (HNPCC) is is one of the most common inherited predispositions to cancer, one of the most common inherited predispositions to cancer, affecting as many as 1 in 500 people in the Western world.affecting as many as 1 in 500 people in the Western world.

Most HNPCC results from a defect in genes that encode the Most HNPCC results from a defect in genes that encode the human counterparts (and homologs) of the bacterial MutS and human counterparts (and homologs) of the bacterial MutS and MutL proteins. The inheritance of HNPCC is autosomal MutL proteins. The inheritance of HNPCC is autosomal dominant. Cells with one functional copy of the mismatch dominant. Cells with one functional copy of the mismatch repair genes have normal mismatch repair activity, but tumor repair genes have normal mismatch repair activity, but tumor cell lines arise from cells that have lost the one functional cell lines arise from cells that have lost the one functional copy and are thus mismatch repair deficient. These cells copy and are thus mismatch repair deficient. These cells display high mutation rates that eventually result in tumor display high mutation rates that eventually result in tumor growth and proliferation. growth and proliferation.

Mismatch repair in humans. (1) Mispairs and misaligned bases Mismatch repair in humans. (1) Mispairs and misaligned bases arise in the course of replication. (2) The G–T-binding protein arise in the course of replication. (2) The G–T-binding protein (GTBP) and the human MutS homolog (hMSH2) recognize the (GTBP) and the human MutS homolog (hMSH2) recognize the incorrect matches. (3) Two additional proteins, hPMS2 and incorrect matches. (3) Two additional proteins, hPMS2 and hMLH1, are recruited and form a larger repair complex. (4) The hMLH1, are recruited and form a larger repair complex. (4) The mismatch is repaired after removal, DNA synthesis, and ligation.mismatch is repaired after removal, DNA synthesis, and ligation.


SOS repairDNA damage often results ina replication block, because DNA synthesis will not DNA damage often results ina replication block, because DNA synthesis will not proceed past a base that cannot specify its complementary partner by hydrogen proceed past a base that cannot specify its complementary partner by hydrogen bonding. In bacterial cells, such replication blocks can be bonding. In bacterial cells, such replication blocks can be bypassedbypassed by inserting by inserting nonspecific bases. The process requires the activation of a special system, the nonspecific bases. The process requires the activation of a special system, the SOS SOS system. system. The name SOS comes from the idea that this system is induced as an The name SOS comes from the idea that this system is induced as an emergency response to prevent cell death in the presence of significant DNA damage. emergency response to prevent cell death in the presence of significant DNA damage. SOS induction is a last resort, allowing the cell to trade death for a certain level of SOS induction is a last resort, allowing the cell to trade death for a certain level of mutagenesis. mutagenesis.

The The recArecA gene, takes part in postreplication gene, takes part in postreplication repair. Here the DNA replication system stalls repair. Here the DNA replication system stalls at a UV photodimer and then restarts past the at a UV photodimer and then restarts past the block, leaving a single-stranded gap. This block, leaving a single-stranded gap. This process leads to few errors.process leads to few errors.

SOS bypass, in contrast, is highly mutagenic. SOS bypass, in contrast, is highly mutagenic. Here the replication system continues past the Here the replication system continues past the lesion, accepting noncomplementary lesion, accepting noncomplementary nucleotides for new strand synthesisnucleotides for new strand synthesis


Schemes for postreplication repair(a) In recombinational repair, replication jumps across a blocking lesion, leaving a gap (a) In recombinational repair, replication jumps across a blocking lesion, leaving a gap in the new strand. A in the new strand. A recArecA-directed protein then fills the gap, using a piece from the -directed protein then fills the gap, using a piece from the opposite parental strand (because of DNA complemen-tarity, this filler will supply the opposite parental strand (because of DNA complemen-tarity, this filler will supply the correct bases for the gap). Finally, the RecA protein repairs the gap in the parental correct bases for the gap). Finally, the RecA protein repairs the gap in the parental strand. strand.

(b) In SOS bypass, when (b) In SOS bypass, when replication reaches a blocking replication reaches a blocking lesion, the SOS system inserts lesion, the SOS system inserts the necessary number of bases the necessary number of bases (often incorrect ones) directly (often incorrect ones) directly across from the lesion and across from the lesion and replication continues without a replication continues without a gap. Note that with either gap. Note that with either pathway the original blocking pathway the original blocking lesion is still there and must be lesion is still there and must be repaired by some other repair repaired by some other repair pathway. pathway.


Summary of repair mechanisms We can now assess the overall repair system strategy used by the cell. It We can now assess the overall repair system strategy used by the cell. It

would be convenient if enzymes could be used to directly reverse each would be convenient if enzymes could be used to directly reverse each specific lesion. However, sometimes that is not chemically possible, and not specific lesion. However, sometimes that is not chemically possible, and not every possible type of DNA damage can be anticipated.every possible type of DNA damage can be anticipated.

Therefore, a general excision repair system is used to remove any type of Therefore, a general excision repair system is used to remove any type of damaged base that causes a recognizable distortion in the double helix.damaged base that causes a recognizable distortion in the double helix.

When lesions are too subtle to cause such a distortion, specific excision When lesions are too subtle to cause such a distortion, specific excision systems, glycosylases, or removal systems are designed.systems, glycosylases, or removal systems are designed.

To eliminate replication errors, a postreplication mismatch repair system To eliminate replication errors, a postreplication mismatch repair system operates; finally, postreplication recombinational systems eliminate gaps operates; finally, postreplication recombinational systems eliminate gaps across from blocking lesions that have escaped the other repair systems.across from blocking lesions that have escaped the other repair systems.

The SOS system is the last resort for a cell to survive a potentially lethald The SOS system is the last resort for a cell to survive a potentially lethald DNA damageDNA damage


DNA repair and mutation rates The repair processes are so efficient that the observed base substitution rate is as The repair processes are so efficient that the observed base substitution rate is as

low as 10low as 10−10−10 to 10 to 10−9−9 per base pair per cell per generation in per base pair per cell per generation in E. coli.E. coli. However, mutant However, mutant strains with increased spontaneous mutation rates have been detected. Such strains strains with increased spontaneous mutation rates have been detected. Such strains are termed are termed mutatorsmutators.. In many cases, the mutator phenotype is due to a defective In many cases, the mutator phenotype is due to a defective repair system. In humans, these repair defects often lead to serious diseases.repair system. In humans, these repair defects often lead to serious diseases.

In In E. coli,E. coli, the mutator loci the mutator loci mutH, mutL, mutU,mutH, mutL, mutU, and and mutSmutS affect components of the affect components of the postreplication mismatch repair system, as does the postreplication mismatch repair system, as does the damdam locus, which specifies the locus, which specifies the enzyme deoxyadenosine methylase. Strains that are Dam− cannot methylate enzyme deoxyadenosine methylase. Strains that are Dam− cannot methylate adenines at GATC sequences, and so the mismatch repair system can no longer adenines at GATC sequences, and so the mismatch repair system can no longer discriminate between the template and the newly synthesized strands. This failure to discriminate between the template and the newly synthesized strands. This failure to discriminate leads to a higher spontaneous mutation rate.discriminate leads to a higher spontaneous mutation rate.

Mutations in the Mutations in the mutYmutY locus result in GC → TA transversions, because many G–A locus result in GC → TA transversions, because many G–A mispairs and all 8-oxodG–A mispairs are unrepaired. The mispairs and all 8-oxodG–A mispairs are unrepaired. The mutMmutM gene encodes a gene encodes a glycosylase that removes 8-oxodG. Strains lacking glycosylase that removes 8-oxodG. Strains lacking mutMmutM are mutators for the GC are mutators for the GC → TA transversion. Strains that are MutT− have elevated rates of the AT → CG → TA transversion. Strains that are MutT− have elevated rates of the AT → CG transversion, because they lack an activity that prevents the incorporation of 8-transversion, because they lack an activity that prevents the incorporation of 8-oxodG across from adenine.oxodG across from adenine.

Strains that are Ung− are missing the enzyme uracil DNA glycosylase. These Strains that are Ung− are missing the enzyme uracil DNA glycosylase. These mutants cannot excise the uracil resulting from cytosine deaminations and, as a mutants cannot excise the uracil resulting from cytosine deaminations and, as a result, have elevated levels of C → T transitions. The result, have elevated levels of C → T transitions. The mutDmutD locus is responsible for locus is responsible for a very high rate of mutagenesis (at least three orders of magnitude higher than a very high rate of mutagenesis (at least three orders of magnitude higher than normal). Mutations at this locus affect the proofreading functions of DNA normal). Mutations at this locus affect the proofreading functions of DNA polymerase III.polymerase III.

genetica per scienze naturali a.a. 05-06 prof s. presciuttini biological repair mechanisms there are...

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