Which Of The Following Mechanisms Normally Repairs Pyrimidine Dimers?

DNA, like whatever other molecule, can undergo a variety of chemic reactions. Because Deoxyribonucleic acid uniquely serves as a permanent re-create of the cell genome, however, changes in its structure are of much greater event than are alterations in other cell components, such every bit RNAs or proteins. Mutations can result from the incorporation of incorrect bases during Deoxyribonucleic acid replication. In add-on, various chemical changes occur in DNA either spontaneously (Figure v.19) or as a result of exposure to chemicals or radiation (Figure 5.twenty). Such impairment to DNA tin cake replication or transcription, and can event in a high frequency of mutations—consequences that are unacceptable from the standpoint of prison cell reproduction. To maintain the integrity of their genomes, cells accept therefore had to evolve mechanisms to repair damaged Deoxyribonucleic acid. These mechanisms of DNA repair can exist divided into two general classes: (ane) direct reversal of the chemic reaction responsible for Deoxyribonucleic acid damage, and (2) removal of the damaged bases followed by their replacement with newly synthesized DNA. Where Deoxyribonucleic acid repair fails, additional mechanisms take evolved to enable cells to cope with the harm.

Figure 5.19

Spontaneous damage to Deoxyribonucleic acid. In that location are two major forms of spontaneous Deoxyribonucleic acid damage: (A) deamination of adenine, cytosine, and guanine, and (B) depurination (loss of purine bases) resulting from cleavage of the bond betwixt the purine bases and deoxyribose, (more...)

Figure 5.xx

Examples of Dna damage induced by radiation and chemicals. (A) UV lite induces the formation of pyrimidine dimers, in which two adjacent pyrimidines (e.g., thymines) are joined by a cyclobutane ring structure. (B) Alkylation is the add-on of methyl (more...)

Direct Reversal of DNA Damage

Most damage to Deoxyribonucleic acid is repaired past removal of the damaged bases followed by resynthesis of the excised region. Some lesions in Deoxyribonucleic acid, nonetheless, tin can be repaired by straight reversal of the damage, which may be a more efficient way of dealing with specific types of DNA harm that occur frequently. Only a few types of DNA damage are repaired in this way, particularly pyrimidine dimers resulting from exposure to ultraviolet (UV) lite and alkylated guanine residues that have been modified by the addition of methyl or ethyl groups at the O⁶ position of the purine ring.

UV light is 1 of the major sources of damage to DNA and is as well the well-nigh thoroughly studied class of DNA damage in terms of repair mechanisms. Its importance is illustrated by the fact that exposure to solar UV irradiation is the cause of almost all pare cancer in humans. The major type of damage induced past UV light is the formation of pyrimidine dimers, in which adjacent pyrimidines on the same strand of DNA are joined by the formation of a cyclobutane ring resulting from saturation of the double bonds betwixt carbons 5 and 6 (see Figure 5.20A). The germination of such dimers distorts the structure of the Deoxyribonucleic acid concatenation and blocks transcription or replication by the site of damage, then their repair is closely correlated with the power of cells to survive UV irradiation. 1 mechanism of repairing UV-induced pyrimidine dimers is direct reversal of the dimerization reaction. The procedure is called photoreactivation because energy derived from visible light is utilized to pause the cyclobutane ring structure (Figure v.21). The original pyrimidine bases remain in DNA, now restored to their normal state. As might be expected from the fact that solar UV irradiation is a major source of DNA harm for various cell types, the repair of pyrimidine dimers past photoreactivation is common to a diversity of prokaryotic and eukaryotic cells, including E. coli, yeasts, and some species of plants and animals. Curiously, however, photoreactivation is non universal; many species (including humans) lack this mechanism of Dna repair.

Effigy five.21

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

Another grade 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 of operations, thereby chemically modifying the base of operations (meet Figure v.20B). A particularly important type of damage is methylation of the O^vi position of guanine, considering the product, O⁶-methylguanine, forms complementary base pairs with thymine instead of cytosine. This lesion tin be repaired by an enzyme (called O^six-methylguanine methyltransferase) that transfers the methyl grouping from O⁶-methylguanine to a cysteine remainder in its agile site (Figure 5.22). The potentially mutagenic chemical modification is thus removed, and the original guanine is restored. Enzymes that catalyze this straight repair reaction are widespread in both prokaryotes and eukaryotes, including humans.

Figure 5.22

Repair of O⁶-methylguanine. O^vi-methylguanine methyltransferase transfers the methyl group from O⁶-methylguanine to a cysteine rest in the enzyme'south active site.

Excision Repair

Although direct repair is an efficient way of dealing with item types of Dna impairment, excision repair is a more than general means of repairing a wide diverseness of chemical alterations to DNA. Consequently, the various types of excision repair are the most important DNA repair mechanisms in both prokaryotic and eukaryotic cells. In excision repair, the damaged Dna is recognized and removed, either as costless bases or as nucleotides. The resulting gap is then filled in by synthesis of a new Deoxyribonucleic acid strand, using the undamaged complementary strand every bit 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 good example of base-excision repair, in which unmarried damaged bases are recognized and removed from the DNA molecule (Figure 5.23). Uracil tin arise in DNA by two mechanisms: (1) Uracil (as dUTP [deoxyuridine triphosphate]) is occasionally incorporated in place of thymine during DNA synthesis, and (ii) uracil can be formed in DNA by the deamination of cytosine (run into Figure 5.19A). The second mechanism is of much greater biological significance because it alters the normal pattern of complementary base of operations 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 bond linking the base (uracil) to the deoxyribose of the Deoxyribonucleic acid backbone. This reaction yields costless uracil and an apyrimidinic site—a sugar with no base of operations 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 by oxidation or ionizing radiation.

Figure five.23

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

The issue of Deoxyribonucleic acid glycosylase action is the formation of an apyridiminic or apurinic site (generally called an AP site) in DNA. Like AP sites are formed as the result of the spontaneous loss of purine bases (see Figure v.19B), which occurs at a significant rate under normal cellular conditions. For example, each cell in the human body is estimated to lose several thousand purine bases daily. These sites are repaired by AP endonuclease, which cleaves adjacent to the AP site (see Effigy 5.23). The remaining deoxyribose moiety is so removed, and the resulting single-base of operations gap is filled by DNA polymerase and ligase.

Whereas DNA glycosylases recognize merely specific forms of damaged bases, other excision repair systems recognize a wide variety of damaged bases that distort the Deoxyribonucleic acid molecule, including UV-induced pyrimidine dimers and bulky groups added to Deoxyribonucleic acid bases as a result of the reaction of many carcinogens with DNA (see Figure 5.20C). This widespread class of Dna repair is known every bit nucleotide-excision repair, because the damaged bases (e.chiliad., a thymine dimer) are removed equally role of an oligonucleotide containing the lesion (Figure 5.24).

Figure 5.24

Nucleotide-excision repair of thymine dimers. Damaged DNA is recognized and then broken 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 Due east. coli, nucleotide-excision repair is catalyzed by the products of three genes (uvrA, B, and C) that were identified because mutations at these loci result in extreme sensitivity to UV low-cal. The protein UvrA recognizes damaged DNA and recruits UvrB and UvrC to the site of the lesion. UvrB and UvrC then cleave on the iii′ and five′ sides of the damaged site, respectively, thus excising an oligonucleotide consisting of 12 or xiii bases. The UvrABC circuitous is often called an excinuclease, a name that reflects its ability to directly excise an oligonucleotide. The activeness of a helicase is then required to remove the damage-containing oligonucleotide from the double-stranded Deoxyribonucleic acid molecule, and the resulting gap is filled by Dna polymerase I and sealed by ligase.

Nucleotide-excision repair systems have also been studied extensively in eukaryotes, particularly in yeasts and in humans. In yeasts, equally in Eastward. coli, several genes involved in Deoxyribonucleic acid repair (called RAD genes for radiation sensitivity) have been identified past the isolation of mutants with increased sensitivity to UV light. In humans, DNA repair genes have been identified largely by studies of individuals suffering from inherited diseases resulting from deficiencies in the ability to repair DNA damage. The nigh extensively studied of these diseases is xeroderma pigmentosum (XP), a rare genetic disorder that affects approximately one in 250,000 people. Individuals with this disease 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 key discovery that cultured cells from XP patients were deficient in the ability to carry out nucleotide-excision repair. This observation not but provided the commencement link between Dna repair and cancer, but also suggested the use of XP cells as an experimental system to identify human Dna repair genes. The identification of man DNA repair genes has been accomplished by studies not only of XP cells, but also of ii other human diseases resulting from DNA repair defects (Cockayne's syndrome and trichothiodystrophy) and of UV-sensitive mutants of rodent prison cell lines. The availability of mammalian cells with defects in DNA repair has allowed the cloning of repair genes based on the ability of wild-type alleles to restore normal UV sensitivity to mutant cells in cistron transfer assays, thereby opening the door to experimental analysis of nucleotide-excision repair in mammalian cells.

Molecular cloning has at present identified 7 dissimilar repair genes (designated XPA through XPG) that are mutated in cases of xeroderma pigmentosum, every bit well as in some cases of Cockayne'due south syndrome, trichothiodystrophy, and UV-sensitive mutants of rodent cells. Table 5.1 lists the enzymes encoded by these genes. Some UV-sensitive rodent cells take mutations in yet another repair gene, chosen ERCC1 (for due eastxcision repair cross complementing), which has not been found to be mutated in known human diseases. Information technology is notable that the proteins encoded by these man DNA repair genes are closely related to proteins encoded by yeast RAD genes, indicating that nucleotide-excision repair is highly conserved throughout eukaryotes.

Table v.1

Enzymes Involved in Nucleotide-Excision Repair.

With cloned yeast and human being repair genes bachelor, information technology has been possible to purify their encoded proteins and develop in vitro systems to written report 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 possibly also XPC) initiates repair past recognizing damaged Deoxyribonucleic acid and forming complexes with other proteins involved in the repair process. These include the XPB and XPD proteins, which act as helicases that unwind the damaged DNA. In add-on, the binding of XPA to damaged DNA 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 DNA on the 5′ and iii′ sides of the damaged site, respectively. This cleavage excises an oligonucleotide consisting of approximately xxx bases. The resulting gap then appears to be filled in past Dna polymerase δ or ε (in association with RFC and PCNA) and sealed by ligase.

An intriguing feature of nucleotide-excision repair is its 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 harm 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 machinery of transcription-repair coupling involves recognition of RNA polymerase stalled at a lesion in the Deoxyribonucleic acid strand being transcribed. The stalled RNA polymerase is recognized past a protein called transcription-repair coupling cistron, which displaces RNA polymerase and recruits the UvrABC excinuclease to the site of damage.

Although the molecular mechanism of transcription-repair coupling in mammalian cells is not yet known, it is noteworthy that the XPB and XPD helicases are components of a multisubunit transcription cistron (called TFIIH) that is required to initiate the transcription of eukaryotic genes (encounter Affiliate six). Thus, these helicases appear to exist 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 besides characterized from a failure to preferentially repair transcribed Deoxyribonucleic acid strands, suggesting that the proteins encoded by the two genes known to be responsible for this disease (CSA and CSB) role in transcription-coupled repair. In improver, 1 of the genes responsible for inherited breast cancer in humans (BRCA1) appears to encode a protein specifically involved in transcription-coupled repair of oxidative Deoxyribonucleic acid harm, suggesting that defects in this type of Deoxyribonucleic acid repair can lead to the evolution of one of the about common cancers in women.

A tertiary excision repair arrangement recognizes mismatched bases that are incorporated during DNA replication. Many such mismatched bases are removed by the proofreading activeness of Dna polymerase. The ones that are missed are subject to later on correction past the mismatch repair system, which scans newly replicated Deoxyribonucleic acid. If a mismatch is found, the enzymes of this repair system are able to identify and excise the mismatched base specifically from the newly replicated Deoxyribonucleic acid strand, allowing the fault to be corrected and the original sequence restored.

In East. coli, the power of the mismatch repair organization to distinguish between parental Dna and newly synthesized DNA is based on the fact that DNA of this bacterium is modified by the methylation of adenine residues within the sequence GATC to grade 6-methyladenine (Figure 5.25). Since methylation occurs later replication, newly synthesized Deoxyribonucleic acid strands are not methylated and thus can exist 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 deed together with an exonuclease and a helicase to excise the DNA between the strand break and the mismatch, with the resulting gap being filled by DNA polymerase and ligase.

Figure 5.25

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

Eukaryotes have a like mismatch repair system, although the mechanism by which eukaryotic cells identify 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 by the presence of unmarried-strand breaks (which would be nowadays in newly replicated DNA) in the strand to be repaired (Figure 5.26). The eukaryotic homologs of MutS and MutL so bind to the mismatched base of operations and direct excision of the Dna between the strand interruption and the mismatch, as in Due east. coli. The importance of this repair system is dramatically illustrated by the fact that mutations in the human homologs of MutS and MutL are responsible for a mutual type of inherited colon cancer (hereditary nonpolyposis colorectal cancer, or HNPCC). HNPCC is one of the most common inherited diseases; it affects every bit many as 1 in 200 people and is responsible for almost 15% of all colorectal cancers in this country. The relationship between HNPCC and defects in mismatch repair was discovered in 1993, when ii groups of researchers cloned the human homolog of MutS and found that mutations in this factor were responsible for about one-half of all HNPCC cases. Subsequent studies have shown that most of the remaining cases of HNPCC are acquired by mutations in one of three man genes that are homologs of MutL.

Figure 5.26

Mismatch repair in mammalian cells. Mismatch repair in mammalian cells is like to E. coli, except that the newly replicated strand is distinguished from the parental strand because information technology contains strand breaks. MutS and MutL demark to the mismatched base of operations (more...)

Postreplication Repair

The direct reversal and excision repair systems deed to correct DNA damage earlier replication, and so that replicative DNA synthesis can proceed using an undamaged Deoxyribonucleic acid strand as a template. Should these systems fail, 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 by the normal activity of Dna polymerases, so replication is blocked at the sites of such damage. Downstream of the damaged site, however, replication can be initiated once more past the synthesis of an Okazaki fragment and can proceed along the damaged template strand (Figure 5.27). The result is a daughter strand that has a gap opposite the site of damage to the parental strand. One of two types of mechanisms may be used to repair such gaps in newly synthesized DNA: recombinational repair or error-prone repair.

Effigy 5.27

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

Recombinational repair depends on the fact that one strand of the parental DNA was undamaged and therefore was copied during replication to yield a normal daughter molecule (see Figure 5.27). The undamaged parental strand can be used to fill the gap contrary the site of damage in the other girl molecule by recombination betwixt homologous Dna sequences (run into the adjacent section). Because the resulting gap in the previously intact parental strand is reverse an undamaged strand, it tin can exist filled in by DNA polymerase. Although the other parent molecule still retains the original damage (e.yard., a pyrimidine dimer), the harm at present lies opposite a normal strand and tin can be dealt with after by excision repair. By a similar mechanism, recombination with an intact DNA molecule tin can be used to repair double strand breaks, which are frequently introduced into DNA by radiation and other dissentious agents.

In error-prone repair, a gap opposite a site of Deoxyribonucleic acid damage is filled past newly synthesized DNA. Since the new Deoxyribonucleic acid is synthesized from a damaged template strand, this form of DNA synthesis is very inaccurate and leads to frequent mutations. It is used simply in bacteria that have been subjected to potentially lethal conditions, such equally all-encompassing 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 cell division and induction of repair systems to cope with a high level of Deoxyribonucleic acid harm. Under these weather, fault-prone repair mechanisms are used, presumably as a way of dealing with damage and so all-encompassing that cell expiry is the only alternative.

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Molecular Medicine : Colon Cancer and DNA Repair.