If no new mutations occur, it would be most reasonable

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Genomes are dynamic entities that change over time as a result of the cumulative effects of small-scale sequence alterations caused by mutation and larger scale rearrangements arising from recombination. Mutation and recombination can both be defined as processes that result in changes to a genome, but they are unrelated and we must make a clear distinction between them:

Both mutation and recombination can have dramatic effects on the cell in which they occur. A mutation in a key gene may cause the cell to die if the protein coded by the mutant gene is defective (Section 14.1.2), and some recombination events lead to defining changes in the biochemical capabilities of the cell, for example by determining the mating type of a yeast cell or the immunological properties of a mammalian B or T lymphocyte. Other mutation and recombination events have a less significant impact on the phenotype of the cell and many have none at all. As we will see in Chapter 15, all events that are not lethal have the potential to contribute to the evolution of the genome but for this to happen they must be inherited when the organism reproduces. With a single-celled organism such as a bacterium or yeast, all genome alterations that are not lethal or reversible are inherited by daughter cells and become permanent features of the lineage that descends from the original cell in which the alteration occurred. In a multicellular organism, only those events that occur in germ cells are relevant to genome evolution. Changes to the genomes of somatic cells are unimportant in an evolutionary sense, but they will have biological relevance if they result in a deleterious phenotype that affects the health of the organism.

14.2. DNA Repair

In view of the thousands of damage events that genomes suffer every day, coupled with the errors that occur when the genome replicates, it is essential that cells possess efficient repair systems. Without these repair systems a genome would not be able to maintain its essential cellular functions for more than a few hours before key genes became inactivated by DNA damage. Similarly, cell lineages would accumulate replication errors at such a rate that their genomes would become dysfunctional after a few cell divisions.

Most cells possess four different categories of DNA repair system (; Lindahl and Wood, 1999):

  • Direct repair systems (Section 14.2.1), as the name suggests, act directly on damaged nucleotides, converting each one back to its original structure.

  • Mismatch repair (Section 14.2.3) corrects errors of replication, again by excising a stretch of single-stranded DNA containing the offending nucleotide and then repairing the resulting gap.

Figure 14.18

Four categories of DNA repair system. See the text for details.

Most if not all organisms also possess systems that enable them to replicate damaged regions of their genome without prior repair. We will examine these systems in Section 14.2.5, and in Section 14.2.6 we will survey the human diseases that result from defects in DNA repair processes.

14.2.1. Direct repair systems fill in nicks and correct some types of nucleotide modification

Most of the types of DNA damage that are caused by chemical or physical mutagens (Section 14.1.1) can only be repaired by excision of the damaged nucleotide followed by resynthesis of a new stretch of DNA, as shown in . Only a few types of damaged nucleotide can be repaired directly:

  • Nicks can be repaired by a DNA ligase if all that has happened is that a phosphodiester bond has been broken, without damage to the 5′-phosphate and 3′-hydroxyl groups of the nucleotides either side of the nick (). This is often the case with nicks resulting from the effects of ionizing radiation.

  • Some forms of alkylation damage are directly reversible by enzymes that transfer the alkyl group from the nucleotide to their own polypeptide chains. Enzymes capable of doing this are known in many different organisms and include the Ada enzyme of E. coli, which is involved in an adaptive process that this bacterium is able to activate in response to DNA damage. Ada removes alkyl groups attached to the oxygen groups at positions 4 and 6 of thymine and guanine, respectively, and can also repair phosphodiester bonds that have become methylated. Other alkylation repair enzymes have more restricted specificities, an example being human MGMT (O6-methylguanine-DNA methyltransferase) which, as its name suggests, only removes alkyl groups from position 6 of guanine.

  • Cyclobutyl dimers are repaired by a light-dependent direct system called photoreactivation. In E. coli, the process involves the enzyme called DNA photolyase (more correctly named deoxyribodipyrimidine photolyase). When stimulated by light with a wavelength between 300 and 500 nm the enzyme binds to cyclobutyl dimers and converts them back to the original monomeric nucleotides. Photoreactivation is a widespread but not universal type of repair: it is known in many but not all bacteria and also in quite a few eukaryotes, including some vertebrates, but is absent in humans and other placental mammals. A similar type of photoreactivation involves the (6-4) photoproduct photolyase and results in repair of (6-4) lesions. Neither E. coli nor humans have this enzyme but it is possessed by a variety of other organisms.

14.2.2. Excision repair

The direct types of damage reversal described above are important, but they form a very minor component of the DNA repair mechanisms of most organisms. This point is illustrated by the draft human genome sequences, which appear to contain just a single gene coding for a protein involved in direct repair (the MGMT gene), but which have at least 40 genes for components of the excision repair pathways (Wood et al., 2001). These pathways fall into two categories:

  • Base excision repair involves removal of a damaged nucleotide base, excision of a short piece of the polynucleotide around the AP site thus created, and resynthesis with a DNA polymerase.

  • Nucleotide excision repair is similar to base excision repair but is not preceded by removal of a damaged base and can act on more substantially damaged areas of DNA.

We will examine each of these pathways in turn.

Base excision repairs many types of damaged nucleotide

Base excision is the least complex of the various repair systems that involve removal of one or more damaged nucleotides followed by resynthesis of DNA to span the resulting gap. It is used to repair many modified nucleotides whose bases have suffered relatively minor damage resulting from, for example, exposure to alkylating agents or ionizing radiation (Section 14.1.1). The process is initiated by a DNA glycosylase which cleaves the β-N-glycosidic bond between a damaged base and the sugar component of the nucleotide (). Each DNA glycosylase has a limited specificity (), the specificities of the glycosylases possessed by a cell determining the range of damaged nucleotides that can be repaired by the base excision pathway. Most organisms are able to deal with deaminated bases such as uracil (deaminated cytosine) and hypoxanthine (deaminated adenine), oxidation products such as 5-hydroxycytosine and thymine glycol, and methylated bases such as 3-methyladenine, 7-methylguanine and 2-methylcytosine (Seeberg et al., 1995). Other DNA glycosylases remove normal bases as part of the mismatch repair system (Section 14.2.3). Most of the DNA glycosylases involved in base excision repair are thought to diffuse along the minor groove of the DNA double helix in search of damaged nucleotides, but some may be associated with the replication enzymes.

Figure 14.20

Base excision repair. (A) Excision of a damaged nucleotide by a DNA glycosylase. (B) Schematic representation of the base excision repair pathway. Alternative versions of the pathway are described in the text.

A DNA glycosylase removes a damaged base by ‘flipping’ the structure to a position outside of the helix and then detaching it from the polynucleotide (Kunkel and Wilson, 1996; Roberts and Cheng, 1998). This creates an AP or baseless site (see ) which is converted into a single nucleotide gap in the second step of the repair pathway (). This step can be carried out in a variety of ways. The standard method makes use of an AP endonuclease, such as exonuclease III or endonuclease IV of E. coli or human APE1, which cuts the phosphodiester bond on the 5′ side of the AP site. Some AP endonucleases can also remove the sugar from the AP site, this being all that remains of the damaged nucleotide, but others lack this ability and so work in conjunction with a separate phosphodiesterase. An alternative pathway for converting the AP site into a gap utilizes the endonuclease activity possessed by some DNA glycosylases, which can make a cut at the 3′ side of the AP site, probably at the same time that the damaged base is removed, followed again by removal of the sugar by a phosphodiesterase.

The single nucleotide gap is filled by a DNA polymerase, using base-paring with the undamaged base in the other strand of the DNA molecule to ensure that the correct nucleotide is inserted. In E. coli the gap is filled by DNA polymerase I and in mammals by DNA polymerase β (see Table 13.2; Sobol et al., 1996). Yeast seems to be unusual in that it uses its main DNA replicating enzyme, DNA polymerase δ, for this purpose (Seeberg et al., 1995). After gap filling, the final phosphodiester bond is put in place by a DNA ligase.

Nucleotide excision repair is used to correct more extensive types of damage

Nucleotide excision repair has a much broader specificity than the base excision system and is able to deal with more extreme forms of damage such as intrastrand crosslinks and bases that have become modified by attachment of large chemical groups. It is also able to correct cyclobutyl dimers by a dark repair process, providing those organisms that do not have the photoreactivation system (such as humans) with a means of repairing this type of damage.

In nucleotide excision repair, a segment of single-stranded DNA containing the damaged nucleotide(s) is excised and replaced with new DNA. The process is therefore similar to base excision repair except that it is not preceded by selective base removal, and a longer stretch of polynucleotide is excised. The best studied example of nucleotide excision repair is the short patch process of E. coli, so called because the region of polynucleotide that is excised and subsequently ‘patched’ is relatively short, usually 12 nucleotides in length.

Short patch repair is initiated by a multienzyme complex called the UvrABC endonuclease, sometimes also referred to as the ‘excinuclease’. In the first stage of the process a trimer comprising two UvrA proteins and one copy of UvrB attaches to the DNA at the damaged site. How the site is recognized is not known but the broad specificity of the process indicates that individual types of damage are not directly detected and that the complex must search for a more general attribute of DNA damage such as distortion of the double helix. UvrA may be the part of the complex most involved in damage location because it dissociates once the site has been found and plays no further part in the repair process. Departure of UvrA allows UvrC to bind (), forming a UvrBC dimer that cuts the polynucleotide either side of the damaged site. The first cut is made by UvrB at the fifth phosphodiester bond downstream of the damaged nucleotide, and the second cut is made by UvrC at the eighth phosphodiester bond upstream, resulting in the 12 nucleotide excision, although there is some variability, especially in the position of the UvrB cut site. The excised segment is then removed, usually as an intact oligonucleotide, by DNA helicase II, which presumably detaches the segment by breaking the base pairs holding it to the second strand. UvrC also detaches at this stage, but UvrB remains in place and bridges the gap produced by the excision. The bound UvrB is thought to prevent the single-stranded region that has been exposed from base-pairing with itself, but alternative roles could be to prevent this strand from becoming damaged, or possibly to direct the DNA polymerase to the site that needs to be repaired. As in base excision repair, the gap is filled by DNA polymerase I and the last phosphodiester bond is synthesized by DNA ligase.

Figure 14.21

Short patch nucleotide excision repair in Escherichia coli. The damaged nucleotide is shown distorting the helix because this is thought to be one of the recognition signals for the UvrAB trimer that initiates the short patch process. See the text for (more...)

E. coli also has a long patch nucleotide excision repair system that involves Uvr proteins but differs in that the piece of DNA that is excised can be anything up to 2 kb in length. Long patch repair has been less well studied and the process is not understood in detail, but it is presumed to work on more extensive forms of damage, possibly regions where groups of nucleotides, rather than just individual ones, have become modified. The eukaryotic nucleotide excision repair process is also called ‘long patch’ but results in replacement of only 24–29 nucleotides of DNA. In fact, there is no ‘short patch’ system in eukaryotes and the name is used to distinguish the process from base excision repair. The system is more complex than in E. coli and the relevant enzymes do not seem to be homologs of the Uvr proteins. In humans at least 16 proteins are involved, with the downstream cut being made at the same position as in E. coli - the fifth phosphodiester bond - but with a more distant upstream cut, resulting in the longer excision. Both cuts are made by endonucleases that attack single-stranded DNA specifically at its junction with a double-stranded region, indicating that before the cuts are made the DNA around the damage site has been melted, presumably by a helicase (). This activity is provided at least in part by TFIIH, one of the components of the RNA polymerase II initiation complex (see Table 9.5). At first it was assumed that TFIIH simply had a dual role in the cell, functioning separately in both transcription and repair, but now it is thought that there is a more direct link between the two processes (Lehmann, 1995; Svejstrup et al., 1996). This view is supported by the discovery of transcription-coupled repair, which repairs some forms of damage in the template strands of genes that are being actively transcribed. The first type of transcriptioncoupled repair to be discovered was a modified version of nucleotide excision, but it now known that base-excision repair is also coupled with transcription (Cooper et al., 1997). These discoveries do not imply that nontranscribed regions of the genome are not repaired. The excision repair processes protect the entire genome from damage, but it is entirely logical that special mechanisms should exist for directing the processes at genes that are being transcribed. The template strands of these genes contain the genome's biological information and maintaining their integrity should be the highest priority for the repair systems.

Figure 14.22

Outline of the events involved during nucleotide excision repair in eukaryotes. The endonucleases that remove the damaged region make cuts specifically at the junction between single-stranded and double-stranded regions of a DNA molecule. The DNA is therefore (more...)

14.2.3. Mismatch repair: correcting errors of replication

Each of the repair systems that we have looked at so far - direct, base excision and nucleotide excision repair - recognize and act upon DNA damage caused by mutagens. This means that they search for abnormal chemical structures such as modified nucleotides, cyclobutyl dimers and intrastrand crosslinks. They cannot, however, correct mismatches resulting from errors in replication because the mismatched nucleotide is not abnormal in any way, it is simply an A, C, G or T that has been inserted at the wrong position. As these nucleotides look exactly like any other nucleotide, the mismatch repair system that corrects replication errors has to detect not the mismatched nucleotide itself but the absence of base-pairing between the parent and daughter strands. Once it has found a mismatch, the repair system excises part of the daughter polynucleotide and fills in the gap, in a manner similar to base and nucleotide excision repair.

The scheme described above leaves one important question unanswered. The repair must be made in the daughter polynucleotide because it is in this newly synthesized strand that the error has occurred; the parent polynucleotide has the correct sequence. How does the repair process know which strand is which? In E. coli the answer is that the daughter strand is, at this stage, undermethylated and can therefore be distinguished from the parent polynucleotide, which has a full complement of methyl groups. E. coli DNA is methylated because of the activities of the DNA adenine methylase (Dam), which converts adenines to 6-methyladenines in the sequence 5′-GATC-3′, and the DNA cytosine methylase (Dcm), which converts cytosines to 5-methylcytosines in 5′-CCAGG-3′ and 5′-CCTGG-3′. These methylations are not mutagenic, the modified nucleotides having the same base-pairing properties as the unmodified versions. There is a delay between DNA replication and methylation of the daughter strand, and it is during this window of opportunity that the repair system scans the DNA for mismatches and makes the required corrections in the undermethylated, daughter strand ().

Figure 14.23

Methylation of newly synthesized DNA in Escherichia coli does not occur immediately after replication, providing a window of opportunity for the mismatch repair proteins to recognize the daughter strands and correct replication errors.

E. coli has at least three mismatch repair systems, called ‘long patch’, ‘short patch and ‘very short patch’, the names indicating the relative lengths of the excised and resynthesized segments. The long patch system replaces up to a kb or more of DNA and requires the MutH, MutL and MutS proteins, as well as the DNA helicase II that we met during nucleotide excision repair. MutS recognizes the mismatch and MutH distinguishes the two strands by binding to unmethylated 5′-GATC-3′ sequences (). The role of MutL is unclear but it might coordinate the activities of the other two proteins so that MutH binds to 5′-GATC-3′ sequences only in the vicinity of mismatch sites recognized by MutS. After binding, MutH cuts the phosphodiester bond immediately upstream of the G in the methylation sequence and DNA helicase II detaches the single strand. There does not appear to be an enzyme that cuts the strand downstream of the mismatch; instead the detached single-stranded region is degraded by an exonuclease that follows the helicase and continues beyond the mismatch site. The gap is then filled in by DNA polymerase I and DNA ligase. Similar events are thought to occur during short and very short mismatch repair, the difference being the specificities of the proteins that recognize the mismatch. The short patch system, which results in excision of a segment less than 10 nucleotides in length, begins when MutY recognizes an A-G or A-C mismatch, and the very short repair system corrects G-T mismatches which are recognized by the Vsr endonuclease.

Figure 14.24

Long patch mismatch repair in Escherichia coli. See the text for details.

Eukaryotes have homologs of the E. coli Mut proteins and their mismatch repair processes probably work in a similar way (Kolodner, 2000). The one difference is that methylation might not be the method used to distinguish between the parent and daughter polynucleotides. Methylation has been implicated in mismatch repair in mammalian cells, but the DNA of some eukaryotes, including fruit flies and yeast, is not extensively methylated; it is thought that these organisms must therefore use a different method. Possibilities include an association between the repair enzymes and the replication complex, so that repair is coupled with DNA synthesis, or use of single-strand binding proteins that mark the parent strand.

14.2.4. Repair of double-stranded DNA breaks

A single-stranded break in a double-stranded DNA molecule, such as is produced during the base and nucleotide excision repair processes and by some types of oxidative damage, does not present the cell with a critical problem. The double helix retains its overall intactness and the break can be repaired by template-dependent DNA synthesis (). A double-stranded break is more serious because this converts the original double helix into two separate fragments which have to be brought back together again in order for the break to be repaired (). The two broken ends must be protected from further degradation, which could result in a deletion mutation appearing at the repaired break point. The repair processes must also ensure that the correct ends are joined: if there are two broken chromosomes in the nucleus, then the correct pairs must be brought together so that the original structures are restored. Experimental studies of mouse cells indicate that achieving this outcome is difficult and if two chromosomes are broken then misrepair resulting in hybrid structures occurs relatively frequently (Richardson and Jasin, 2000). Even if only one chromosome is broken, there is still a possibility that a natural chromosome end could be confused as a break and an incorrect repair made. This type of error is not unknown, despite the presence of special telomerebinding proteins that mark the natural ends of chromosomes (Section 2.2.1).

Figure 14.25

Single- and double-strand-break repair. (A) A single-strand break does not disrupt the integrity of the double helix. The exposed single strand is coated with PARP1 proteins, which protect this intact strand from breaking and prevent it from participating (more...)

Double-strand breaks are generated by exposure to ionizing radiation and some chemical mutagens, and are also made by the cell, in a controlled fashion, during recombination events such as the genome rearrangements that join together immunoglobulin gene segments and T-cell receptor gene segments in B and T lymphocytes (Section 12.2.1). Progress in understanding the break repair system has been stimulated by studies of mutant human cell lines, which have resulted in the identification of various sets of genes involved in the process (Critchlow and Jackson, 1998). These genes specify a multi-component protein complex that directs a DNA ligase to the break (). The complex includes a protein called Ku, made up of two non-identical subunits, which binds the DNA ends either side of the break (Walker et al., 2001). Ku binds to the DNA in association with the DNA-PKCS protein kinase, which activates a third protein, XRCC4, which interacts with the mammalian DNA ligase IV, directing this repair protein to the double-strand break.

Figure 14.26

Non-homologous end-joining (NHEJ) in humans. (A) The repair process. Additional proteins not shown in the diagram are also involved in NHEJ. These include the protein kinases ATM and ATR (Section 13.3.2), whose main role may be to signal to the cell the (more...)

The repair process is called non-homologous endjoining (NHEJ), the name indicating that there is no need for homology between the two molecules whose ends are being joined, unlike other end-joining mechanisms that we will encounter when we study recombination in Section 14.3. NHEJ is looked on as a type of recombination because, as well as repairing breaks, it can be used to join molecules or fragments that were not previously joined, producing new combinations. A version of the NHEJ system is probably used during construction of immunoglobulin and T-cell receptor genes, but the details are likely to be different because these programmed rearrangements of the genome involve intermediate structures, such as DNA hairpin loops, that are not seen during the repair of DNA breaks resulting from damage.

14.2.5. Bypassing DNA damage during genome replication

If a region of the genome has suffered extensive damage then it is conceivable that the repair processes will be overwhelmed. The cell then faces a stark choice between dying or attempting to replicate the damaged region even though this replication may be error-prone and result in mutated daughter molecules. When faced with this choice E. coli cells invariably take the second option, by inducing one of several emergency procedures for bypassing sites of major damage. The best studied of these bypass processes is the SOS response, which enables the cell to replicate its DNA even though the template polynucleotides contain AP sites and/or cyclobutyl dimers and other photoproducts resulting from exposure to chemical mutagens or UV radiation that would normally block, or at least delay, the replication complex. Bypass of these sites requires construction of a mutasome, comprising the UmuD′2C complex (also called DNA polymerase V, a trimer made up of two UmuD′ proteins and one copy of UmuC) and several copies of the RecA protein (Goodman, 2000). The latter is a single-stranded DNA-binding protein that coats the damaged strands, enabling the UmuD′2C complex to displace DNA polymerase III and carry out error-prone DNA synthesis until the damaged region has been passed and DNA polymerase III can take over once again ().

Figure 14.27

The SOS response of Escherichia coli. See the text for details.

The SOS response is primarily looked on as the last best chance that the bacterium has to replicate its DNA and hence survive under adverse conditions. However, the price of survival is an increased mutation rate because the mutasome does not repair damage, it simply allows a damaged region of a polynucleotide to be replicated. When it encounters a damaged position in the template DNA, the polymerase selects a nucleotide more or less at random, although with some preference for placing an A opposite an AP site: in effect the error rate of the replication process increases. It has been suggested that this increased mutation rate is the purpose of the SOS response, mutation being in some way an advantageous response to DNA damage, but this idea remains controversial (Chicurel, 2001).

For some time, the SOS response was thought to be the only damage-bypass process in bacteria, but we now appreciate that at least two other E. coli polymerases act in a similar way, although with different types of damage. These are DNA polymerase II, which can bypass nucleotides bound to mutagenic chemicals such as N-2-acetylaminofluorene, and DNA polymerase IV (also called DinB), which can replicate through a region of template DNA in which the two parent polynucleotides have become misaligned (Lindahl and Wood, 1999; Hanaoka, 2001). Bypass polymerases have also been discovered in eukaryotic cells. These include DNA polymerase η, which can bypass cyclobutyl dimers (Johnson et al., 1999), and DNA polymerases ι and ζ, which work together to replicate through photoproducts and AP sites (Johnson et al., 2000).

14.2.6. Defects in DNA repair underlie human diseases, including cancers

The importance of DNA repair is emphasized by the number and severity of inherited human diseases that have been linked with defects in one of the repair processes. One of the best characterized of these is xeroderma pigmentosum, which results from a mutation in any one of several genes for proteins involved in nucleotide excision repair. Nucleotide excision is the only way in which human cells can repair cyclobutyl dimers and other photoproducts, so it is no surprise that the symptoms of xeroderma pigmentosum include hypersensitivity to UV radiation, patients suffering more mutations than normal on exposure to sunlight, which often leads to skin cancer (Lehmann, 1995). Trichothiodystrophy is also caused by defects in nucleotide excision repair, but this is a more complex disorder which, although not involving cancer, usually includes problems with both the skin and nervous system.

A few diseases have been linked with defects in the transcription-coupled component of nucleotide excision repair. These include breast and ovarian cancers, the BRCA1 gene that confers susceptibility to these cancers coding for a protein that has been implicated, at least indirectly, with transcription-coupled repair (Gowen et al., 1998), and Cockayne syndrome, a complex disease manifested by growth and neurologic disorders (Hanawalt, 2000). A deficiency in transcription-coupled repair has also been identified in humans suffering from the cancer-susceptibility syndrome called HNPCC (hereditary non-polyposis colorectal cancer; Mellon et al., 1996), although this disease was originally identified as a defect in mismatch repair (Kolodner, 1995). Ataxia telangiectasia, the symptoms of which include sensitivity to ionizing radiation, results from defects in the ATX gene, which is involved in the damage-detection process (Section 13.3.2). Other diseases that are associated with a breakdown in DNA repair are Bloom's and Werner's syndromes, which are caused by inactivation of a DNA helicase that may have a role in NHEJ (Shen and Loeb, 2000; Wu and Hickson, 2001), and Fanconi's anemia, which confers sensitivity to chemicals that cause crosslinks in DNA but whose biochemical basis is not yet known.

Is it reasonable to state that mutations are essential to the evolutionary process?

Mutations are essential to evolution; they are the raw material of genetic variation. Without mutation, evolution could not occur.

Which of the following is a true statement about mutations?

Answer and Explanation: The correct answer to this question is B. They always cause a change to an organism's genotype. Regardless of the cause of the change, a DNA sequence is always altered when it mutates.

Is a random mutation more likely to be beneficial or harmful?

By the same token , any random change in a gene's DNA is likely to result in a protein that does not function normally or may not function at all. Such mutations are likely to be harmful. Harmful mutations may cause genetic disorders or cancer. A genetic disorder is a disease caused by a mutation in one or a few genes.

Is mutation necessary for evolution to occur Why?

Mutation is important as the first step of evolution because it creates a new DNA sequence for a particular gene, creating a new allele. Recombination also can create a new DNA sequence (a new allele) for a specific gene through intragenic recombination.