Which Of The Following Repair Mechanisms Involves Direct Reversal Of Damaged Dna?

DNA, like whatever other molecule, can undergo a diversity of chemical reactions. Considering DNA uniquely serves as a permanent copy of the cell genome, nonetheless, changes in its structure are of much greater consequence than are alterations in other prison cell components, such every bit RNAs or proteins. Mutations tin can result from the incorporation of incorrect bases during Deoxyribonucleic acid replication. In improver, diverse chemical changes occur in Dna either spontaneously (Effigy 5.19) or as a issue of exposure to chemicals or radiations (Figure 5.xx). Such damage to Dna tin can block replication or transcription, and can result in a loftier frequency of mutations—consequences that are unacceptable from the standpoint of cell reproduction. To maintain the integrity of their genomes, cells accept therefore had to evolve mechanisms to repair damaged Dna. These mechanisms of DNA repair can be divided into ii full general classes: (ane) directly reversal of the chemical reaction responsible for DNA damage, and (2) removal of the damaged bases followed by their replacement with newly synthesized Dna. Where DNA repair fails, additional mechanisms have evolved to enable cells to cope with the harm.

Figure 5.xix

Spontaneous damage to DNA. There are two major forms of spontaneous DNA damage: (A) deamination of adenine, cytosine, and guanine, and (B) depurination (loss of purine bases) resulting from cleavage of the bond between the purine bases and deoxyribose, (more...)

Effigy 5.xx

Examples of Deoxyribonucleic acid damage induced by radiation and chemicals. (A) UV light induces the formation of pyrimidine dimers, in which ii adjacent pyrimidines (e.g., thymines) are joined by a cyclobutane ring structure. (B) Alkylation is the addition of methyl (more...)

Direct Reversal of DNA Impairment

Most damage to DNA is repaired by removal of the damaged bases followed by resynthesis of the excised region. Some lesions in DNA, nevertheless, can be repaired past direct reversal of the damage, which may be a more efficient way of dealing with specific types of Deoxyribonucleic acid damage that occur frequently. Only a few types of Deoxyribonucleic acid harm are repaired in this way, peculiarly pyrimidine dimers resulting from exposure to ultraviolet (UV) lite and alkylated guanine residues that have been modified past the addition of methyl or ethyl groups at the O^{half dozen} position of the purine ring.

UV light is one of the major sources of impairment to DNA and is also the almost thoroughly studied form of Deoxyribonucleic acid damage in terms of repair mechanisms. Its importance is illustrated by the fact that exposure to solar UV irradiation is the crusade of virtually all skin cancer in humans. The major type of impairment induced by UV light is the formation of pyrimidine dimers, in which adjacent pyrimidines on the same strand of Dna are joined by the germination of a cyclobutane ring resulting from saturation of the double bonds betwixt carbons 5 and six (run across Effigy five.20A). The germination of such dimers distorts the structure of the DNA chain and blocks transcription or replication by the site of damage, then their repair is closely correlated with the ability of cells to survive UV irradiation. Ane mechanism of repairing UV-induced pyrimidine dimers is direct reversal of the dimerization reaction. The process is called photoreactivation considering energy derived from visible light is utilized to break the cyclobutane band construction (Figure five.21). The original pyrimidine bases remain in DNA, now restored to their normal country. Every bit might be expected from the fact that solar UV irradiation is a major source of Dna damage for diverse cell types, the repair of pyrimidine dimers by photoreactivation is common to a variety of prokaryotic and eukaryotic cells, including E. coli, yeasts, and some species of plants and animals. Curiously, nevertheless, photoreactivation is not universal; many species (including humans) lack this mechanism of Deoxyribonucleic acid repair.

Figure v.21

Direct repair of thymine dimers. UV-induced thymine dimers can be repaired by photoreactivation, in which free energy from visible light is used to dissever the bonds forming the cyclobutane ring.

Another form of direct repair deals with damage resulting from the reaction between alkylating agents and DNA. Alkylating agents are reactive compounds that can transfer methyl or ethyl groups to a Deoxyribonucleic acid base, thereby chemically modifying the base (see Effigy 5.20B). A particularly important blazon of impairment is methylation of the O^six position of guanine, considering the production, O⁶-methylguanine, forms complementary base pairs with thymine instead of cytosine. This lesion can be repaired by an enzyme (called O^{half dozen}-methylguanine methyltransferase) that transfers the methyl grouping from O^half-dozen-methylguanine to a cysteine residue in its active site (Effigy 5.22). The potentially mutagenic chemical modification is thus removed, and the original guanine is restored. Enzymes that catalyze this directly repair reaction are widespread in both prokaryotes and eukaryotes, including humans.

Figure 5.22

Repair of O^half-dozen-methylguanine. O⁶-methylguanine methyltransferase transfers the methyl group from O⁶-methylguanine to a cysteine residue in the enzyme's active site.

Excision Repair

Although direct repair is an efficient mode of dealing with particular types of Dna damage, excision repair is a more general means of repairing a wide variety of chemical alterations to DNA. Consequently, the diverse types of excision repair are the nearly of import DNA repair mechanisms in both prokaryotic and eukaryotic cells. In excision repair, the damaged DNA is recognized and removed, either as free bases or as nucleotides. The resulting gap is then filled in by synthesis of a new Deoxyribonucleic acid strand, using the undamaged complementary strand as a template. Three types of excision repair—base-excision repair, nucleotide-excision repair, and mismatch repair—enable cells to cope with a variety of different kinds of DNA harm.

The repair of uracil-containing DNA is a skilful example of base of operations-excision repair, in which single damaged bases are recognized and removed from the DNA molecule (Figure 5.23). Uracil can ascend in Dna by two mechanisms: (1) Uracil (as dUTP [deoxyuridine triphosphate]) is occasionally incorporated in place of thymine during Dna synthesis, and (2) uracil can be formed in DNA past the deamination of cytosine (encounter Effigy 5.19A). The second mechanism is of much greater biological significance because it alters the normal pattern of complementary base pairing and thus represents a mutagenic event. The excision of uracil in Deoxyribonucleic acid is catalyzed by Deoxyribonucleic acid glycosylase, an enzyme that cleaves the bail linking the base (uracil) to the deoxyribose of the DNA courage. This reaction yields complimentary uracil and an apyrimidinic site—a carbohydrate with no base attached. DNA glycosylases also recognize and remove other abnormal bases, including hypoxanthine formed by the deamination of adenine, pyrimidine dimers, alkylated purines other than O⁶-alkylguanine, and bases damaged past oxidation or ionizing radiation.

Figure v.23

Base of operations-excision repair. In this case, uracil (U) has been formed by deamination of cytosine (C) and is therefore contrary a guanine (G) in the complementary strand of DNA. The bond between uracil and the deoxyribose is broken by a DNA glycosylase, leaving (more...)

The result of DNA glycosylase action is the formation of an apyridiminic or apurinic site (more often than not called an AP site) in Dna. Like AP sites are formed equally the result of the spontaneous loss of purine bases (see Effigy 5.19B), which occurs at a significant charge per unit under normal cellular weather condition. For example, each cell in the human torso is estimated to lose several k purine bases daily. These sites are repaired by AP endonuclease, which cleaves side by side to the AP site (see Figure five.23). The remaining deoxyribose moiety is and then removed, and the resulting single-base gap is filled by Deoxyribonucleic acid polymerase and ligase.

Whereas Dna glycosylases recognize merely specific forms of damaged bases, other excision repair systems recognize a broad variety of damaged bases that distort the DNA molecule, including UV-induced pyrimidine dimers and beefy groups added to DNA bases as a event of the reaction of many carcinogens with Deoxyribonucleic acid (see Figure 5.20C). This widespread form of DNA repair is known as nucleotide-excision repair, considering the damaged bases (e.g., a thymine dimer) are removed as part of an oligonucleotide containing the lesion (Effigy v.24).

Figure five.24

Nucleotide-excision repair of thymine dimers. Damaged DNA is recognized and then cleaved on both sides of a thymine dimer by iii′ and 5′ nucleases. Unwinding by a helicase results in excision of an oligonucleotide containing the damaged (more...)

In Eastward. coli, nucleotide-excision repair is catalyzed by the products of iii genes (uvrA, B, and C) that were identified because mutations at these loci result in extreme sensitivity to UV light. The poly peptide UvrA recognizes damaged DNA and recruits UvrB and UvrC to the site of the lesion. UvrB and UvrC and then cleave on the 3′ and 5′ sides of the damaged site, respectively, thus excising an oligonucleotide consisting of 12 or 13 bases. The UvrABC complex is often called an excinuclease, a name that reflects its power to straight excise an oligonucleotide. The action of a helicase is then required to remove the damage-containing oligonucleotide from the double-stranded DNA molecule, and the resulting gap is filled past Dna polymerase I and sealed past ligase.

Nucleotide-excision repair systems take also been studied extensively in eukaryotes, especially in yeasts and in humans. In yeasts, as in Eastward. coli, several genes involved in DNA repair (called RAD genes for radiation sensitivity) have been identified by the isolation of mutants with increased sensitivity to UV calorie-free. In humans, Dna repair genes accept been identified largely by studies of individuals suffering from inherited diseases resulting from deficiencies in the ability to repair DNA damage. The almost extensively studied of these diseases is xeroderma pigmentosum (XP), a rare genetic disorder that affects approximately one in 250,000 people. Individuals with this illness are extremely sensitive to UV light and develop multiple skin cancers on the regions of their bodies that are exposed to sunlight. In 1968 James Cleaver made the central discovery that cultured cells from XP patients were deficient in the ability to acquit out nucleotide-excision repair. This observation not but provided the first link between DNA repair and cancer, but also suggested the use of XP cells every bit an experimental organisation to identify homo Deoxyribonucleic acid repair genes. The identification of man DNA repair genes has been accomplished by studies not but of XP cells, merely besides of two other human diseases resulting from DNA repair defects (Cockayne's syndrome and trichothiodystrophy) and of UV-sensitive mutants of rodent cell lines. The availability of mammalian cells with defects in DNA repair has immune the cloning of repair genes based on the power of wild-blazon alleles to restore normal UV sensitivity to mutant cells in gene transfer assays, thereby opening the door to experimental assay of nucleotide-excision repair in mammalian cells.

Molecular cloning has at present identified vii dissimilar repair genes (designated XPA through XPG) that are mutated in cases of xeroderma pigmentosum, every bit well every bit in some cases of Cockayne's syndrome, trichothiodystrophy, and UV-sensitive mutants of rodent cells. Table 5.1 lists the enzymes encoded by these genes. Some UV-sensitive rodent cells have mutations in yet another repair factor, chosen ERCC1 (for excision repair cross complementing), which has not been plant to be mutated in known human diseases. It is notable that the proteins encoded by these human Deoxyribonucleic acid repair genes are closely related to proteins encoded past yeast RAD genes, indicating that nucleotide-excision repair is highly conserved throughout eukaryotes.

Tabular array five.1

Enzymes Involved in Nucleotide-Excision Repair.

With cloned yeast and human repair genes available, information technology has been possible to purify their encoded proteins and develop in vitro systems to study the repair process. Although some steps remain to be fully elucidated, these studies have led to the development of a basic model for nucleotide-excision repair in eukaryotic cells. In mammalian cells, the XPA protein (and peradventure also XPC) initiates repair past recognizing damaged Dna and forming complexes with other proteins involved in the repair process. These include the XPB and XPD proteins, which act every bit helicases that unwind the damaged DNA. In addition, the binding of XPA to damaged Deoxyribonucleic acid leads to the recruitment of XPF (as a heterodimer with ERCC1) and XPG to the repair complex. XPF/ERCC1 and XPG are endonucleases, which cleave Deoxyribonucleic acid on the 5′ and three′ sides of the damaged site, respectively. This cleavage excises an oligonucleotide consisting of approximately 30 bases. The resulting gap then appears to be filled in by Deoxyribonucleic acid polymerase δ or ε (in association with RFC and PCNA) and sealed by ligase.

An intriguing characteristic of nucleotide-excision repair is its human relationship to transcription. A connection between transcription and repair was first suggested by experiments showing that transcribed strands of DNA are repaired more than rapidly than nontranscribed strands in both E. coli and mammalian cells. Since Dna damage blocks transcription, this transcription-repair coupling is thought to be advantageous by allowing the cell to preferentially repair damage to actively expressed genes. In E. coli, the mechanism of transcription-repair coupling involves recognition of RNA polymerase stalled at a lesion in the Dna strand existence transcribed. The stalled RNA polymerase is recognized by a poly peptide chosen transcription-repair coupling gene, which displaces RNA polymerase and recruits the UvrABC excinuclease to the site of impairment.

Although the molecular mechanism of transcription-repair coupling in mammalian cells is not still known, it is noteworthy that the XPB and XPD helicases are components of a multisubunit transcription factor (called TFIIH) that is required to initiate the transcription of eukaryotic genes (see Affiliate 6). Thus, these helicases appear to be required for the unwinding of DNA during both transcription and nucleotide-excision repair, providing a direct biochemical link between these two processes. Patients suffering from Cockayne's syndrome are also characterized from a failure to preferentially repair transcribed Dna strands, suggesting that the proteins encoded by the 2 genes known to be responsible for this disease (CSA and CSB) office in transcription-coupled repair. In addition, ane of the genes responsible for inherited breast cancer in humans (BRCA1) appears to encode a protein specifically involved in transcription-coupled repair of oxidative Dna harm, suggesting that defects in this type of DNA repair tin lead to the development of one of the most common cancers in women.

A third excision repair organization recognizes mismatched bases that are incorporated during Dna replication. Many such mismatched bases are removed by the proofreading activity of DNA polymerase. The ones that are missed are subject to afterwards correction by the mismatch repair system, which scans newly replicated Dna. If a mismatch is establish, the enzymes of this repair system are able to identify and excise the mismatched base specifically from the newly replicated Dna strand, allowing the error to be corrected and the original sequence restored.

In E. coli, the ability of the mismatch repair organisation to distinguish between parental DNA and newly synthesized Deoxyribonucleic acid is based on the fact that Dna of this bacterium is modified by the methylation of adenine residues within the sequence GATC to course 6-methyladenine (Effigy v.25). Since methylation occurs afterwards replication, newly synthesized DNA strands are not methylated and thus can be specifically recognized by the mismatch repair enzymes. Mismatch repair is initiated by the protein MutS, which recognizes the mismatch and forms a complex with two other proteins chosen MutL and MutH. The MutH endonuclease then cleaves the unmethylated Dna strand at a GATC sequence. MutL and MutS then act together with an exonuclease and a helicase to excise the Dna betwixt the strand break and the mismatch, with the resulting gap being filled by DNA polymerase and ligase.

Figure v.25

Mismatch repair in E. coli. The mismatch repair system detects and excises mismatched bases in newly replicated DNA, which is distinguished from the parental strand because it has not yet been methylated. MutS binds to the mismatched base of operations, followed past (more than...)

Eukaryotes have a like mismatch repair organisation, although the mechanism by which eukaryotic cells place newly replicated Dna differs from that used by E. coli. In mammalian cells, information technology appears that the strand-specificity of mismatch repair is determined past the presence of unmarried-strand breaks (which would exist nowadays in newly replicated DNA) in the strand to be repaired (Figure five.26). The eukaryotic homologs of MutS and MutL so demark to the mismatched base of operations and direct excision of the Deoxyribonucleic acid betwixt the strand break and the mismatch, as in Due east. coli. The importance of this repair arrangement is dramatically illustrated past the fact that mutations in the human homologs of MutS and MutL are responsible for a common type of inherited colon cancer (hereditary nonpolyposis colorectal cancer, or HNPCC). HNPCC is ane of the most mutual inherited diseases; it affects equally many as 1 in 200 people and is responsible for virtually fifteen% of all colorectal cancers in this country. The relationship between HNPCC and defects in mismatch repair was discovered in 1993, when two groups of researchers cloned the human homolog of MutS and constitute that mutations in this gene were responsible for nearly one-half of all HNPCC cases. Subsequent studies have shown that almost of the remaining cases of HNPCC are caused by mutations in 1 of three human genes that are homologs of MutL.

Figure 5.26

Mismatch repair in mammalian cells. Mismatch repair in mammalian cells is like to Eastward. coli, except that the newly replicated strand is distinguished from the parental strand because it contains strand breaks. MutS and MutL bind to the mismatched base (more...)

Postreplication Repair

The direct reversal and excision repair systems act to correct DNA damage before replication, so that replicative Deoxyribonucleic acid synthesis can go on using an undamaged Deoxyribonucleic acid strand as a template. Should these systems neglect, however, the cell has alternative mechanisms for dealing with damaged DNA at the replication fork. Pyrimidine dimers and many other types of lesions cannot be copied past the normal action of Dna polymerases, so replication is blocked at the sites of such damage. Downstream of the damaged site, even so, replication can exist initiated again by the synthesis of an Okazaki fragment and can go on forth the damaged template strand (Effigy v.27). The result is a daughter strand that has a gap contrary the site of damage to the parental strand. 1 of two types of mechanisms may be used to repair such gaps in newly synthesized Dna: recombinational repair or fault-prone repair.

Figure five.27

Postreplication repair. The presence of a thymine dimer blocks replication, but Deoxyribonucleic acid polymerase can bypass the lesion and reinitiate replication at a new site downstream of the dimer. The result is a gap contrary the dimer in the newly synthesized Dna (more...)

Recombinational repair depends on the fact that one strand of the parental Deoxyribonucleic acid was undamaged and therefore was copied during replication to yield a normal daughter molecule (encounter Figure five.27). The undamaged parental strand can be used to fill the gap reverse the site of impairment in the other daughter molecule by recombination between homologous DNA sequences (come across the side by side section). Because the resulting gap in the previously intact parental strand is contrary an undamaged strand, information technology can exist filled in by Deoxyribonucleic acid polymerase. Although the other parent molecule still retains the original impairment (e.g., a pyrimidine dimer), the damage now lies opposite a normal strand and tin can be dealt with later on past excision repair. By a similar machinery, recombination with an intact Dna molecule can be used to repair double strand breaks, which are frequently introduced into Deoxyribonucleic acid by radiation and other damaging agents.

In error-prone repair, a gap reverse a site of DNA damage is filled past newly synthesized Deoxyribonucleic acid. Since the new Dna is synthesized from a damaged template strand, this form of DNA synthesis is very inaccurate and leads to frequent mutations. Information technology is used just in bacteria that accept been subjected to potentially lethal conditions, such as extensive UV irradiation. Such treatments induce the SOS response, which may be viewed as a mechanism for dealing with extreme environmental stress. The SOS response includes inhibition of prison cell partition and induction of repair systems to cope with a loftier level of Dna damage. Under these weather, error-prone repair mechanisms are used, presumably as a mode of dealing with impairment then extensive that jail cell expiry is the only alternative.