In gene mutation, one allele of a gene changes into a different allele. Because such a change takes place within a single gene and maps to one chromosomal locus (“point”), a gene mutation is sometimes called a point mutation. This terminology originated before the advent of DNA sequencing and therefore before it was routinely possible to discover the molecular basis for a mutational event. Mutants are a race of strange creatures found throughout the world and factions of Pox Nora, although almost all of them belong the the wrath factions. This race, like the Beast race, groups a large number of different entities that fall under several themes, and some members show only basic levels of intelligence. Mutants are a race, not a theme, but they do include one full theme in Creepers.

Efforts to understand human physiology through the study of champion athletes and record performances have been ongoing for about a century. For endurance sports three main factors – maximal oxygen consumption, the so-called ‘lactate threshold’ and efficiency (i.e. The oxygen cost to generate a give running speed or cycling power output) – appear to play key roles in endurance performance. And lactate threshold interact to determine the ‘performance ‘ which is the oxygen consumption that can be sustained for a given period of time. Efficiency interacts with the performance to establish the speed or power that can be generated at this oxygen consumption.

This review focuses on what is currently known about how these factors interact, their utility as predictors of elite performance, and areas where there is relatively less information to guide current thinking. In this context, definitive ideas about the physiological determinants of running and cycling efficiency is relatively lacking in comparison with and the lactate threshold, and there is surprisingly limited and clear information about the genetic factors that might pre-dispose for elite performance. It should also be cautioned that complex motivational and sociological factors also play important roles in who does or does not become a champion and these factors go far beyond simple physiological explanations. Therefore, the performance of elite athletes is likely to defy the types of easy explanations sought by scientific reductionism and remain an important puzzle for those interested in physiological integration well into the future. IntroductionFaster, Higher, Stronger: these simple descriptions have been of interest to humans since the beginning of recorded history. In this context, integrative physiology has long been served by so-called ‘experiments in nature.’ These include asking fundamental questions about the ability of various animal species to function in harsh environments and studies on unique human patients with clinical conditions that offer the opportunity to ask important questions about physiological regulation (for examples see;; ).

Along similar lines, studies on both human and animal performance in athletic events can provide important insights and raise critical questions about oxygen transport, muscle performance and metabolism, cardiovascular control, and the operation of various components of the nervous system. Historical noteOne of the first analyses of world records from A. Hill in 1925 related the decline in running speed as race distance increased to the topic of muscle fatigue. Even before then, the Italian physiologist Mosso, who was interested in fatigue associated with manual labour, noted ‘It is not will, not the nerves, but it is the muscle that finds itself worn out after the intense work of the brain.’ But Mosso hedged his bets and also commented that ‘fatigue of brain reduces the strength of the muscles’. Hill's original plot of world record performance time on the X-axis versus performance speed on the Y-axisThe top tracing is for speed-skating, the middle tracing is for running by males, and the bottom tracing is for running by women. The shape of the curve led to Hill's original ideas about differing causes of muscle fatigue for exercise bouts of different durations.In Hill's analysis he speculated that the factors limiting performance in events of less than a minute and more than an hour are probably not dependent solely on the energy supply to the contracting muscles and discussed the physiological determinants of performance in the context of ideas about energy stores, oxygen demand and oxygen debt.

He also speculated that there were ‘three types of fatigue’ (a concept he found to be inexact) including: (1) one associated with short violent efforts; (2) the exhaustion ‘which overcomes the body when an effort of moderate intensity is continued for a long time’; and (3) fatigue associated with a more general ‘wear-and-tear’. The first two types of fatigue were thought to be primarily ‘muscular’.Additionally, for the second and third types of fatigue, which occur during endurance exercise, Hill speculated that as distances increase beyond about 10 miles (∼16 km), ‘The continued fall in the curve, as the effort is prolonged, is probably due to the second and third types of fatigue which we discussed above, either to the exhaustion of the material of the muscle, or to the incidental disturbances which may make a man stop before his muscular system has reached its limit. A man of average size running in a race must expend about 300 g of glycogen per hour; perhaps a half of this may be replaced by its equivalent of fat. After a very few hours therefore the whole glycogen supply of his body will be exhausted.

The body, however, does not readily use fat alone as a source of energy; disturbances may arise in the metabolism; it will be necessary to feed a man with carbohydrate as the effort continues. Such feeding will be followed by digestion; disturbances of digestion may occur – other reactions may ensue. For very long distances the case is far more complex than for the shorter ones, and although, no doubt, the physiological principles can be ascertained, we do not know enough about them yet to be able further to analyse the curves.’ These comments and the work of Scandinavian physiologists in the 1930s set the stage for the concept of carbohydrate loading and a number of dietary and feeding strategies that have been shown to delay fatigue (;; ). Focus of this reviewIn this review for The Journal of Physiology's 2008 Olympic Issue, we will focus on current models of human performance, review the physiological ‘ideas’ that led to these models, and ask what these models explain and more importantly do not explain. Presents our concepts using a model like Hill's that is focused on ‘performance velocity’ and how it is determined by maximum rates of aerobic energy production, anaerobic capacity and how efficiently the energy being used is converted to movement. Overall schematic of the multiple physiological factors that interact as determinants of performance velocity or power outputThis figure serves as the conceptual framework for the ideas discussed in this review.In general, we focus on endurance exercise performance because it is our area of expertise, and there are relatively more data on the physiological adaptations that contribute to endurance performance, and (especially for running) there are accurate records extending for more than 100 years. There is also at least some physiological data on champion athletes over almost the same period of time.

Overview of current ideas about human performanceAs noted above, most models of athletic performance focus on distance running and endurance cycling. First, there are excellent records and standard events. Second, there is comprehensive physiological data on a large number of elite athletes. Third, it is possible, using treadmills and cycle ergometers, to reasonably simulate in a laboratory what is happening during actual competition. We should also note that for the purposes of this review we assume that environmental conditions are ideal and do not add any additional challenges to physiological regulation (most notably the challenges associated with high altitude and/or high environmental temperatures).

Several well-accepted concepts (,;; ) have emerged related to endurance exercise performance velocity and the first component issue is the level of aerobic metabolism that can be maintained during a race (i.e. Performance; ). The upper limit for this is ‘maximal’ oxygen uptake. This is usually achieved during relatively large muscle mass exercise and represents the integrative ability of the heart to generate a high cardiac output, total body haemoglobin, high muscle blood flow and muscle oxygen extraction, and in some cases the ability of the lungs to oxygenate the blood (;;;;; ). By the 1930s very high values for in athletes were observed and identified as a marker of elite performance. Champion endurance athletes have values of between 70 and 85 ml kg −1 min −1, with values in women typically averaging about 10% lower due to lower haemoglobin concentrations and higher levels of body fat (;;; ).In summary, values 50–100% greater than those seen in normally active healthy young subjects are seen in champion endurance athletes and the most striking adaptations to training that contribute to these high values include increased cardiac stroke volume, increased blood volume, increased capillary density and mitochondrial density in the trained muscles.

Of these, the most dominant factor is a high stroke volume (;; ).Once it became reasonably clear that elite runners had high values for it also became clear that for events lasting beyond 10 or 15 min, most or all of the competition was performed at an average pace that did not evoke, with much of the 42 km marathon run at approximately 75–85% while 10 km is performed at 90–100% and 5 km at close to (; ). Along these lines, it has recently been shown that maximal aerobic metabolism can decline acutely during the course of a 5–8 min laboratory performance bout. This decline is caused by a fall in stroke volume and accelerated muscle fatigue due to reduced blood and oxygen delivery and increased anaerobic metabolism (; ). This does not invalidate the concept of, but rather indicates that the maximal rate of aerobic ATP resynthesis during a race is dynamic and that truly accurate models of energy turnover during actual competition would require instantaneous measurements and calculation of fluxes through multiple metabolic pathways (e.g. Total ATP turnover with contributions from both aerobic and anaerobic components as well as energy stores). Lactate thresholdBased on the concepts above the question then became what fraction of might be sustained for periods of time extending to several hours (i.e.

The marathon) and what is the rate of glycolysis in the active muscles at this rate of mitochondrial oxidation. This question led to observations showing a curvilinear relationship between blood lactate values during exercise and the distance of the effort (; ) and led to the concept that the rate of aerobic metabolism maintained during a performance bout (i.e. Performance; ) can be better described by the degree of muscle glycolytic stress reflected in lactate production in addition to (; ). Plot or blood lactic acid concentration versus race distance This figure is an example of the diminishing contribution of so-called ‘anaerobic’ energy sources as race distance increases. This paper also set the stage for a number of later investigations related to the fraction of (e.g.

Performance ) that could be sustained in competition.In this context, as running speed or power output on a cycle ergometer increases in untrained subjects there is typically no sustained rise in blood lactate concentration until about 60% of is reached. In trained subjects this value can be 75–90% of.

There is a long history of investigation about what causes this rise in blood lactate levels and also how lactate (and/or hydrogen ion) does or does not contribute to fatigue. For this review the important summary points include: (1) the initial appearance of blood lactate is not synonymous with hypoxia in the skeletal muscle, and (2) the lactate molecule per se does not ‘cause’ muscle fatigue (;; ).

Individual record of treadmill velocity and versus blood lactate concentration in subject capable of breaking 2:30 h for the marathon In untrained subjects the upturn in lactic acid concentrations is seen at about 60% of. Trained subjects can usually exercise at 75–85% of before there is a marked increase in blood lactate concentration. This figure also illustrates the concept of performance and performance velocity.What appears to be occurring is that the maximum rate of fat oxidation is inadequate to meet the ATP demands of muscles contracting at moderate and high intensities. This causes intracellular signalling events to occur which stimulate glycogenolysis and glycolysis and ultimately the rate of pyruvate delivery to the mitochondria progressively exceeds the ability of the mitochondria to oxidize pyruvate and this leads to accelerated generation of lactic acid (;; ). The associated hydrogen ion is then a likely culprit in muscle fatigue and also activates group III and IV skeletal muscle afferents that evoke important cardiovascular and autonomic reflexes.While the physiological determinants of the lactate threshold are extremely complex, they are determined mainly by the oxidative capacity of the skeletal muscle (;;;,). This capacity is highly plastic and can essentially increase more than twofold in the trained skeletal muscle of humans or animals who engage in 20–120 min of training at a requisite intensity (;; ). This more than doubling of oxidative capacity is one of the factors that is linked to the high ‘lactate threshold’ values seen in elite endurance athletes.

As noted above, these elite athletes have values that are 50–100% above those seen in normally active sedentary young people and their lactate threshold occurs at a higher percentage of their. This means that in elite athletes the absolute oxygen consumptions (power output and/or speed) that can be generated for long periods of time before reaching the lactate threshold is essentially doubled allowing sustained running speeds of 20 km h −1 or cycling power outputs of 400 W.Other key factors that reduce muscle fatigability and lactate production during exercise at 85–90%, when only a fraction of the total limb muscle mass is simultaneously recruited, is the quantity of muscle mass that the athlete can recruit to share in sustaining power production. Elite cyclists appear capable of rotating power production through 20–25% more muscle mass throughout a 1 h bout of cycling, thus reducing the relative power production and stress on a given fibre (; ). Additionally, this ‘power sharing’ among fibres would also reduce the glycolytic stress and lactate production per fibre due to more total mitochondrial sharing for a given rate of aerobic metabolism. These factors should operate in a complementary way that reduces the stress per mitochondria and muscle fibre.As exercise extends beyond about 2 h the problem becomes one of fuel availability as (Hill predicted) the glycogen content in skeletal muscle becomes depleted and the modest ability of active muscle to take up glucose from blood (via either the liver or from feeding) can limit the rate of oxidative ATP generation and thus the pace that can be sustained.

In some (but not all) subjects the associated reductions in blood glucose evoke frank symptoms of hypoglycaemia that limit the ability of the individual to continue exercising (;, ). Other highly trained subjects show remarkable resistance to hypoglycaemia and for these athletes muscle glycogen depletion is probably more important. In response to these events, a number of pre-competition dietary strategies and during-exercise energy replacement regimens and products have been developed. When these are used in an optimal manner muscle glycogen stores can be augmented by 40% before exercise, and hypoglycaemia can be avoided with the net effect being that the duration of exercise at about the lactate threshold can be extended by about one-third (from 2 to 3 h to 4 h) (,; ). Performance and anaerobic metabolismWithout practical direct calorimetric methods to measure instantaneous rates of heat and work production during endurance exercise (; ), the best practical estimation of the rates of actual metabolic energy production and ATP turnover is obtained from measures of oxygen consumption (i.e.

Indirect calorimetry) during an endurance performance bout. During marathon running the relative amount of anaerobic metabolism is small yet in events lasting 13–30 min (i.e. 5 and 10 km running), it will be significant, contributing perhaps 10–20% of total ATP turnover. This anaerobic contribution to ATP turnover during endurance performance bouts is noted in and has classically been estimated from measures of post-exercise oxygen consumption and may equal the energy provided by 50–80 ml kg −1 of oxygen uptake. However, the rate at which this energy might be generated and consumed is difficult to estimate in a definitive way.also makes the point that the rate of total ATP turnover during endurance performance reflects the interplay of aerobic and anaerobic metabolism with lactate generation serving to maintain the NAD + needed for continued glycolysis and generation of pyruvate.

An example of this interplay appears to be the influence of high skeletal muscle capillary density, serving to remove or recycle within muscle fatiguing metabolites (e.g. Hydrogen ions). As shown in, exercise time to fatigue at 88% in a population of cyclists ( n = 14, individually numbered) possessing the same (i.e. 4.9 l min −1), as expected, was related to the percentage of at the blood lactate threshold. However, some subjects (see upper line in ) were able to exercise longer than normal (see lower line in ) even when accounting for their lactate threshold (i.e. Subjects 1, 2, 7 and 8 in ). For the most part, these individuals (i.e.

1, 2, 7 and 8) possessed an unusually high muscle capillary density which may have allowed their exercising muscles to better tolerate anaerobic metabolism and lactic acid production. For this reason, indicates that ‘Performance, might be directly influenced by muscle capillary density, independent of its important role in delivering oxygen and reducing diffusion gradients, but also by removing waste products and limiting acidosis in the contracting muscles. Time to fatigue during exercise at 88% of plotted against lactate threshold (LT) in 14 highly trained cyclists and triathletes (data plotted from; )These athletes all had similar values and uniformly high muscle oxidative enzymes. A subgroup of 4 athletes (subjects 1, 2, 7 and 8) with exceptionally high capillary density seemed to ‘overachieve’ in comparison with their lactate threshold values compared with other members of the group.An additional point from is that much remains to be learned about subtle factors that delay or accelerate fatigue during events performed at intensities above 80–90% of.

Small increases in total energy expenditure or reductions in oxygen delivery will have disproportionate effects and accelerate fatigue during very intense exercise. At this time it remains unclear if laboratory tests can detect the subtle adaptations in the very best performers who seem to be able manage their metabolism in a way that permits maximum efficient energy use. EfficiencyThe next factor that makes an important contribution in endurance exercise performance velocity has been termed ‘economy’ or ‘efficiency.’ In the above sections we outlined how and the lactate threshold operate to determine ‘Performance,. The next question then is how much speed or power can be generated for that level of oxygen consumption? The oxygen cost of endurance running (ml kg min −1) at a given speed can vary about 30–40% among individuals (;; ), as shown in. When cycling at a given power output, the oxygen cost and thus gross mechanical efficiency also varies from one person to another, but by a somewhat lesser amount compared with running (i.e.

Regression lines for high, average and low running economy (efficiency) in elite endurance athletes based on values gleaned from a number of sources Since there has been little systematic data collected above ∼18 km h −1 the filled triangles in the figure are individual data from a limited number of champions with exceptional running economy. This figure emphasizes the importance of efficiency among groups of elite performers with relatively uniform and lactate threshold values. It is also of note that the physiological determinants of efficiency (especially for running) are poorly understood.Gross mechanical efficiency when endurance-trained cyclists generate 300 W can vary from 18.5 to 23.5% and it appears that more than one-half of this variability is related to the percentage of type I (slow twitch) muscle fibres of the vastus lateralis muscle. The efficiency with which the chemical energy of ATP hydrolysis is converted to physical work depends greatly on the velocity of sarcomere and muscle fibre shortening. Type I (slow twitch) fibres display greater mechanical efficiency when cycling at cadences of 60–120 r.p.m.

Therefore, it is not surprising that elite endurance cyclists typically possess a higher percentage of type I muscle fibres, given that they are more efficient. Although type I muscle fibres in untrained humans possess higher mitochondrial density compared with type II fibres (fast twitch), it is important to note that with intense interval training, mitochondrial activity can be increased to equally high levels in both fibre types. Thus, with intense endurance training over years, the main functional advantage of type I fibres appears to be efficiency when cycling rather than total oxidative ability, although type I fibre seem to retain a greater ability to oxidize fat.It is also of note that many champion cyclists chose pedal cadences of around 90 r.p.m. This is a cadence that may actually increase whole body oxygen consumption slightly for a given total body power output from the minimum which usually occurs at 50–60 r.p.m.

In a comprehensive engineering/physiology analysis of this problem noted that subjects with higher levels of myosin heavy chain I (MHC I, the predominant myosin in type I fibres) self-selected higher pedal rates and these rates closely matched the rate of peak mechanical efficiency. In this context, they speculated that motor control patterns in these subjects might favour a faster cadence so that relatively low total muscle forces (probably from fatigue-resistant motor units) per pedal stroke could generate the needed power so that the higher force (and more fatigable) motor units could be conserved. On a speculative note, with lower force per contraction there might be less compression of the microcirculation in the active muscle and better distribution of blood flow in a way that is consistent with the concepts presented in.Running is a more complicated movement than cycling in that it elicits more stretch on the muscle prior to contraction and there is more potential to capture mechanical energy in the elastic elements of tissue. However, although there has been long-standing interest in identifying the biomechanical and anatomical factors that allow one person compared with another to expend 30–40% less energy per kilogram of body to move at a given velocity, the aetiology of differences in running economy generally remain a mystery, and biomechanical descriptions of running are not good predictors of running economy (; ).Elite endurance runners typically possess a predominance of type I muscle fibres and it would seem logical that they are more mechanically efficient at the velocities of distance running (;; ). However, running and walking economy has not often been highly correlated with a person's percentage of type I muscle fibres (; ). This agrees with the idea that running economy reflects the interaction of numerous factors including muscle morphology, elastic elements and joint mechanics in the efficient transfer of ATP to running speed.The extent to which cycling efficiency or running economy can be improved with training has also been of long-standing interest. Until recently, it was generally believed that cycling efficiency and running economy did not improve much with training.

At best, running economy might sometimes increase slightly over the course of 2 months when explosive-type weight training is added to an endurance training program (; ). However, the conclusion that efficiency does not change with training was based on cross-sectional comparisons of relatively small numbers of endurance athletes.In this context, there are no comprehensive longitudinal data on groups of endurance athletes followed over several years to determine the trainability of cycling efficiency or running economy.

However, there are at least two cases reporting that running economy can be improved over years of training in elite athletes (; ). In fact, the current world record holder for the women's marathon displayed a remarkable 14% improvement in running economy over the course of 5 years of training. Furthermore, cycling efficiency was observed to increase 8% over the course of 7 years in an elite endurance cyclist. In general, these case reports suggest that muscular efficiency and running economy might indeed improve with continued endurance training at a rate of approximately 1–3% per year. One possible contributing factor is that at least some of the fast myosin in endurance-trained muscle shifts to a different and perhaps more efficient isoform. Additionally, in some models of extreme muscle use there can be a complete conversion of fast twitch to slow twitch muscle fibres, whether this occurs in elite athletes who train for two or more hours per day for many years is not known and it is further not known if such a shift would explain any improvements in efficiency that might occur with years of training.

Integrating current ideas about physiological limiting factorsThe concepts above and in suggest that and lactate threshold interact to determine how long a given rate of aerobic and anaerobic metabolism can be sustained (i.e. Rumble fighter online. Performance ) and efficiency then determines how much speed or power (i.e. Performance velocity) can be achieved at a give amount of energy consumption. These relationships were hinted at by Hill in his 1925 paper and were clearly defined in the period between 1970 and the early 1990s (;;;;; ).

When reasonable estimates of the ‘best’ values ever recorded for these three parameters were used in this equation a predicted optimal marathon time of around 1:45 h emerged. Even when assumptions about wind resistance were added, times well under 2 h seemed possible. In retrospect, one overlooked possibility was that the values used in the estimates came from laboratory studies typically conducted while the subjects ran up a grade of 5–10% and these values may be ∼10% higher than those seen during level running.

However, even if the highest values seen during graded running protocols are not attainable during level running in many people, there are high enough and lactate threshold values that might result in sustainable oxygen uptakes, which in combination with outstanding running economy, would generate a marked improvement in current world record time. These comments reinforce the conclusions of this earlier modelling effort that either there are unknown factors that operate at high speeds that make such time ‘not’ achievable or that for some reason ‘best in class’ values for every factor are unlikely to occur in the same person. Some unanswered questionsIn the context of the ideas above there are a number of fundamental unanswered questions.

We have already highlighted questions about the determinants of efficiency especially for running, and for both running and cycling a key question is how ‘trainable’ this factor is. Additionally, we have discussed several factors beyond mitochondrial content and oxidative enzymes that may permit some athletes to operate for prolonged periods at especially high fractions of their. These factors may also be important in events like long distance cycling and cross country skiing that occur over varied terrain and are not conducted at an even physiological pace. In these competitions there are frequent bursts of more intense near-maximal activity lasting from a few seconds to a few minutes that are followed by periods of relative recovery.A fundamental question is the role genetics plays in the attainment of world class status and truly elite athletic performance.

There are a number of studies showing that key elements of the response to training in sedentary persons is widely variable and has a genetic component. There have also been reports suggesting that common single nucleotide polymorphisms might be over represented in either groups of elite endurance athletes or in sedentary subjects that respond most to training. The most notable example is the idea that I (for insertion) variant of the angiotensin converting enzyme (ACE) gene is over represented among elite endurance athletes.

However, in the largest cohort of elite athletes who have been both rigorously phenotyped and genotyped this association has not been confirmed and to date there are no genetic markers identified in humans that have been clearly shown to be more frequent in elite endurance athletes.Another interesting example relates to the gene encoding for the skeletal muscle isoform of AMP deaminase. There is a common mutation of this gene that may be associated with lower exercise capacity and ‘trainability’ in untrained subjects. Concluding remarksOur concepts about factors that regulate and potentially limit endurance performance are not a radical departure from the intuitive logic introduced by Mosso and Hill. Elite athletic performance involves integration of muscular, cardiovascular and neurological factors that function cooperatively to efficiently transfer the energy from aerobic and anaerobic ATP turnover into velocity and power. The past four decades of research have described in great detail the cardiovascular and muscular factors that govern oxygen delivery to active muscles, oxidative ATP rephosphorylation and markers of metabolic stress. However, little advancement seems to have been made in identifying neurological factors that might alter motor unit recruitment during prolonged exercise in ways that limit fatigue. Although it has become increasingly apparent that muscular efficiency and economy are hugely important, the physiological determinants of running economy remain a mystery while myosin type appears important to cycling efficiency at cadences chosen in competition.The outcome of all Olympic endurance events is decided at intensities above 85% and most require athletes to be relatively fatigue resistant at intensities that stimulate significant anaerobic metabolism.

At this time, the literature contains insufficient data that specifically describe the actual total energy demands of competition, the amount of muscle that is active during competition, and the complex neural patterns by which power and velocity are maintained as fatigue and failure develop in the nervous, cardiovascular and muscular systems. Such data are needed both in absolute and temporal terms. In this context, more work is needed on highly trained athletes performing very intense exercise in real or simulated competitions.

In, one of a gene changes into a different allele. Because sucha change takes place within a single gene and maps to one chromosomal locus(“point”), a gene mutation is sometimes called a. This terminology originated before the adventof sequencing and therefore before it was routinely possible to discover themolecular basis for a mutational event. Nowadays, point mutations typically refer toalterations of single base pairs of DNA or of a small number of adjacent base pairs.In this chapter, we focus on such simple point mutations.The constellation of possible ways in which point mutations could change a wild-typeis very large and varies according to the particular structure and sequence ofthe gene. However, it is always true that mutations that reduce or eliminate genefunction (loss-of-function mutations) are the most abundant class. The reason issimple: it is much easier to break a machine than to alter the way that it works byrandomly changing or removing one of its components. For the same reason, mutationsthat increase or alter the type of activity of the gene or where it is expressed(gain-of-function mutations) are much rarer.

Gene Mutations at the Molecular Level.At the level, there are two main types of point mutational changes:base substitutions and base additions ordeletions. Base substitutions are those mutations in which one base pair isreplaced by another. Base substitutions again can be divided into two subtypes:transitions and transversions. To describe these subtypes, we consider how aalters the sequence on one DNA strand (the complementary change willtake place on the other strand.) is the replacement of a base by the other base of thesame chemical category ( replaced by purine: either A to or G to A;replaced by pyrimidine: either to or T to C). A is the opposite—thereplacement of a base of one chemical category by a base of the other(pyrimidine replaced by purine: C to A, C to G, T to A, T to G; purine replacedby pyrimidine: A to C, A to T, G to C, G to T).

In describing the same changesat the double-stranded level of DNA, we must state both members of a base pair:an example of a would be GC → AT; that of a would beGC → TA.Addition or mutations are actually ofpairs; nevertheless, the convention is to callthem base-pair additions or deletions. The simplest of thesemutations are single-base-pair additions or single-base-pair deletions. Thereare examples in which mutations arise through simultaneous addition or deletionof multiple base pairs at once. As we shall see later in this chapter,mechanisms that selectively produce certain kinds of multiple-base-pairadditions or deletions are the cause of certain human genetic diseases.What are the functional consequences of these different types of point mutations?First, consider what happens when a arises in a polypeptidecoding partof a.

For single-base substitutions, there are several possible outcomes,which are direct consequences of two aspects of the: degeneracy ofthe code and the existence of termination codons. 1.Silent substitutions: the changes one for aninto another codon for that same amino acid. 2.Missense mutations: the for one is replacedby a codon for another amino acid. 3.Nonsense mutations: the for one is replacedby a termination (stop) codon.Silent substitutions never alter the sequence of thechain. The severity of the effect of missense and nonsense mutations on thepolypeptide will differ on a case-by-case basis. For example, if a causes the substitution of a chemically similar amino acid, referred toas a, thenit is likely that the alteration will have a less-severe effect on the protein’sstructure and function. Alternatively, chemically different amino acidsubstitutions, called nonsynonymous substitutions, are more likelyto produce severe changes in protein structure and function.

Nonsense mutationswill lead to the premature termination of. Thus, they have aconsiderable effect on protein function. Linear relation between X-ray dose to which Drosophilamelanogaster were exposed and the percentage ofmutations (mainly sex-linked recessive lethals).Recognize that the distinction between induced and spontaneous is purelyoperational. If we are aware that an organism was mutagenized, thenwe infer that any mutations that arise after this mutagenesis were induced.However, this is not true in an absolute sense. The mechanisms that give rise tospontaneous mutations also are in action in this mutagenized organism.

Inreality, there will always be a subset of mutations recovered after mutagenesisthat are independent of the action of the. The proportion of mutationsthat fall into this subset depends on how potent a mutagen is. The higher therate of induced mutations, the lower the proportion of recovered mutations thatare actually “spontaneous” in origin.Induced and spontaneous mutations arise by generally different mechanisms, sothey will be covered separately. After considering these mechanisms, we shallexplore the subject of biological repair. Without these repairmechanisms, the rate of mutation would be so high that cells would accumulatetoo many mutations to remain viable and capable of reproduction. Thus, themutational events that do occur are those rare events that have somehow beenoverlooked or bypassed by the repair processes. Mechanisms of Mutation InductionWhen we examine the array of mutations induced by different mutagens, we see adistinct specificity that is characteristic of each.

Suchmutational specificity was first noted at therII locus of the bacteriophage T4. Specificity arises froma given mutagen’s “preference” both for a certain type of(for example→  transitions) and for certain mutationalsites called hot spots.Mutagens act through at least three different mechanisms. They canreplace a base in the, alter a base sothat it specifically mispairs with another base, or damage abase so that it can no longer pair with any base under normal conditions. Base replacementSome chemical compounds are sufficiently similar to the normalof that they are occasionally incorporated into DNA in place of normalbases; such compounds are called base analogs. Many of theseanalogs have pairing properties unlike those of the normal bases; thus theycan produce mutations by causing incorrect nucleotides to be inserted during. To understand the action of base analogs, we must firstconsider the natural tendency of bases to assume different forms.All of the bases in can exist in one of several forms, calledtautomers, which are isomers that differ in the positionsof their atoms and in the bonds between the atoms.

The forms are inequilibrium. The keto form of each base is normally found inDNA , whereas theenol forms of the bases are rare. The complementary basepairing of the enols is different from that of the keto forms. Demonstrates the possiblemispairs resulting from tautomeric shifts.

Because of suchmispairing, the enols are one source of rare spontaneousmutations. For example, assume that a in DNA changes into its enolform at the moment at which it is copied in the course of (itchanges back into its keto form soon after). The enol form will bind to anincoming. Hence we can represent the mutagenic process as follows,in which.

is the enol form of guanine. Mispairs can also occur when bases become spontaneously ionized.The 5-bromouracil (5-BU) is an analog of thathas bromine at the carbon-5 position in place of the CH 3 groupfound in thymine. Its mutagenic action is based on enolization andionization. In 5-BU, the bromine atom is not in a position in which it canhydrogen-bond during base pairing, so the keto form of 5-BU pairs with, as would thymine, and this pairing is shown in.

However, the presence of the bromineatom significantly alters the of electrons in the base ring, so5-BU can frequently change to either the enol form or anionized form, the latter of which pairs with. 5-BU causes  →  or AT → GCtransitions in the course of, depending on whether 5-BU has beenenolized or ionized or as an incoming base. Hence the action of 5-BUas a mutagen is due to the fact that the molecule spends more of its time inthe enol or ion form. Alternative pairing possibilities for 5-bromouracil (5-BU). 5-BUis an analog of thymine that can be mistakenly incorporated intoDNA as a base. It has a bromine atom in place of the methylgroup.

(a) In its normal keto state, 5-BU mimics the pairingbehaviorAnother widely used in research is 2-aminopurine(2-AP), which is an analog of that can pair withbut, when protonated, can also mispair with, as shown in. Therefore, when 2-AP isincorporated into by pairing with thymine, it can generate  → transitions by mispairing with cytosine in subsequent replications.

Or, if2-AP is incorporated by mispairing with cytosine, then GC → AT transitionswill result when it pairs with thymine. Genetic studies have shown that2-AP, like 5-BU, is highly specific for transitions. Alkylation-induced specific mispairing.

The alkylation (in thiscase, EMS-generated ethylation) of the O-6position of guanine, as well as the O-4 position of thymine, canlead to direct mispairing with thymine and guanine,respectively, as shown here.The intercalating agents are another important class ofmodifiers. This group of compounds includes, acridineorange, and a class of chemicals termed ICRcompounds.These agents are flat planar molecules that mimic base pairs and are able toslip themselves in (intercalate) between the stackedat the core of the DNA.

In this intercalated position, an agentcan cause single-pair insertions or deletions. Intercalatingagents may also stack between bases in single-stranded DNA; in so doing,they may stabilize bases that are looped out during frameshift formation, asdepicted in the Streisinger model. Base damagelarge number of mutagens damage one or more bases, so no specific basepairing is possible. The result is a block, becausesynthesis will not proceed past a base that cannot specify its complementarypartner by hydrogen bonding.

In bacterial cells, such replication blocks canbe bypassed by inserting nonspecific bases. The processrequires the activation of a special system, the SOS system. The nameSOS comes from the idea that this system is induced asan emergency response to prevent cell death in the presence of significantDNA damage. SOS is a last resort, allowing the cell to trade deathfor a certain level of mutagenesis. DNA polymerase III, shown in blue, stops at a non-coding lesion,such as the TC photodimer shown here, generatingsingle-stranded regions that attract the Ssb protein (darkpurple) and RecA (light purple), which forms filaments. ThepresenceExactly how the SOS bypass system functions is not clear, although inE. Coli it is known to be dependent on at least threegenes, recA (which also has a role in general), umuC, and umuD.

Currentmodels for SOS bypass suggest that the UmuC and UmuD proteins combine withthe polymerase III complex to loosen its otherwise strictspecificity and permit replication past noncoding lesions.shows a model for thebypass system operating after III stalls at a type of damagecalled a – photodimer. Because can restart downstream from thedimer, a single-stranded region of DNA is generated. This attracts thestabilizing protein, called single-stranded-binding protein (Ssb), as wellas the RecA protein, which forms filaments and signals the cell tosynthesize the UmuC and UmuD proteins. The UmuD protein binds to thefilaments and is cleaved by the RecA protein to yield a shortened versiontermed UmuD′, which then recruits the UmuC protein to form a complex thatallows DNA polymerase III to continue past the dimer, adding bases acrossfrom the dimer with a high error frequency (see ).Therefore mutagens that damage specific base-pairing sites are dependent onthe SOS system for their action. The category of SOS-dependent mutagens isimportant, because it includes most -causing agents (carcinogens),such as ultraviolet (UV) light and aflatoxin B 1.How does the SOS system take part in the recovery of mutations aftermutagenesis?

Does the SOS system lower the fidelity of replications somuch (to permit the bypass of noncoding lesions) that manyerrors occur, even for undamaged DNA? If this hypothesis were correct, mostmutations generated by different SOS-dependent mutagens would be similar,rather than specific to each. Most mutations would result from theaction of the SOS system itself on undamaged DNA. The mutagen, then, wouldplay the indirect role of inducing the SOS system. Studies of mutationalspecificity, however, have shown that this is not the case.

Instead, aseries of different SOS-dependent mutagens have markedly differentspecificities. Each mutagen induces a unique of mutations.Therefore, the mutations must be generated in response to specific damagedbase pairs.

The type of differs in many cases. Some of the mostwidely studied lesions include UV photoproducts and apurinic sites.Ultraviolet (UV) light generates a number of photoproducts in. Two different lesions that unite adjacent pyrimidines in the samestrand have been most strongly correlated with mutagenesis. These lesionsare the cyclobutane photodimer and the 6-4 photoproduct ( on the following page).These lesions interfere with normal base pairing; hence, of theSOS system is required for mutagenesis. The insertion of incorrect basesacross from UV photoproducts is at the 3′ position of the dimer, and morefrequently for 5′-CC-3′ and 5′-TC-3′ dimers. The → is themost frequent, but other base substitutions (transversions) andframeshifts also are induced by UV light, as are larger duplications anddeletions.

(a) Structure of a cyclobutane pyrimidine dimer. Ultravioletlight stimulates the formation of a four-membered cyclobutylring (green) between two adjacent pyrimidines on the same DNAstrand by acting on the 5,6 double bonds. (b) Structure of the6-4Aflatoxin B 1 (AFB 1 ) is a powerfuloriginally isolated from fungal-infected peanuts. Aflatoxin forms anaddition product at the N-7 position of. This product leads to the breakage ofthe bond between the base and the sugar, thereby liberating the base andresulting in an apurinic site. Studies with apurinic sites generatedhave demonstrated that the SOS bypass of these sites leads to thepreferential insertion of an across from an apurinic site.

Thispredicts that agents that cause depurination at guanine residues shouldpreferentially induce  →  transversions. For example, with 0 (zero)representing an apurinic site. Genetics In Process 7-1: Salvador Luria and Max Delbruck show thatbacterial mutations are random.Spontaneous mutations are very rare, making it difficult to determine theunderlying mechanisms. How then do we have insight into the processes governing? Even though they are rare, some selective systems allownumerous spontaneous mutations to be obtained and then characterized at themolecular level—for example, their sequences can be determined. From thenature of the sequence changes, inferences can be made about the processes thathave led to the spontaneous mutations.

Errors in DNA replicationMispairing in the course of is a source of spontaneous basesubstitution. (Mispairing was covered earlier in the discussion of 5-BU.)Most mispairing mutations are transitions. This is likely to be because an or mispair does not distort the as much as AG orCT base pairs do. However, transversions also can occur through mispairing.Replication errors can also lead to frameshift mutations. Thesequence surrounding frameshift hot spots was determined in thelysozyme of T4.

These mutations often occur at repeated bases.The Streisinger model proposes that frameshifts arise when loops in single-stranded regions arestabilized by the “slipped mispairing” of repeated sequences duringreplication. Additionally, in the E.

Coli lacI gene,certain hot spots result from repeated sequences, just as predicted by theStreisinger model.depicts the of spontaneous mutations in thelacI gene. Note how one or two mutational sitesdominate the distribution. In lacI, a four-base-pairsequence (CTGG) repeated three times in tandem in the is the causeof the hot spots (for simplicity, only one strand of the DNA isshown). The distribution of 140 spontaneous mutations inlacI. Each occurrence of a point mutationis indicated by a box.

Red boxes designate fast-revertingmutations. Deletions (gold) are represented below.

TheI map is given in terms of the amino acidnumberThe major, represented here by the mutations FS5, FS25,FS45, and FS65, results from the addition ofone extra set of the four bases CTGG. The minor hot spot, represented hereby the mutations FS2 and FS84, resultsfrom the loss of one set of the four bases CTGG.How can we explain these observations? The Streisinger model predicts thatthe frequency of a particular frameshift depends on the number of base pairsthat can form during the slipped mispairing of repeated sequences. Thewild-type sequence shown for the lacI can slip out oneCTGG sequence and stabilize this structure by forming nine base pairs (applythe model in to thesequence shown for lacI).

Whether a or an additionis generated depends on whether the slippage is on the or on thenewly synthesized strand, respectively.Larger deletions (more than a few base pairs) constitute a sizable fractionof spontaneous mutations, as shown in. Most, although not all, of the deletions are ofrepeated sequences.shows the first 12 deletions analyzed at the sequence level.

Furtherstudies have shown that the longer repeats constitute hot spots fordeletions. Duplications of segments of DNA have been observed in manyorganisms. Like deletions, they often occur at sequence repeats. Deletions in lacI. Deletions occurring inS74 and S112 are shown atthe top of the diagram. As indicated by the gold bars, one ofthe sequence repeats (green) and all the intervening DNA hasbeen deleted, leaving one copy of the repeated sequence.

AllmutationsHow do these deletions and duplications form? Several mechanisms couldaccount for their formation.

Deletions may be generated aserrors. For example, an extension of the Streisinger model of slippedmispairing couldexplain why deletions predominate at short repeated sequences.Alternatively, deletions and duplications could be generated bybetween the repeats. Spontaneous lesionsNaturally occurring damage to the, called spontaneouslesions, also can generate mutations. Two of the most frequentspontaneous lesions are depurination and deamination, the former being morecommon.We learned earlier that aflatoxin induces depurination; however,depurination also occurs spontaneously.

Mammalian cell spontaneously losesabout 10,000 purines from its during a 20-hour cellgeneration period at37°. If these lesions were to persist, they would result in significantgenetic damage because, during, the apurinic sites cannotspecify any kind of base, let alone the correct one. However, as mentionedearlier in the chapter, under certain conditions, a base can be insertedacross from an apurinic site, frequently resulting in a.The deamination of yields. Unrepaired uracil residues will pairwith in the course of, resulting in the conversion of a pair into an pair (a GC → AT ). Deaminations at certaincytosine positions have been found to be one type of mutational.sequence analysis of hot spots for GC → AT transitions in thelacI has shown that 5-methylcytosine residues arepresent at the position of each hot spot. (Certain bases in prokaryotes andeukaryotes are normally methylated.) Some of the data from thislacI study are shown in. The height of each bar on the graphrepresents the frequency of mutations at each of a number of sites.

It canbe seen that the positions of 5-methylcytosine residues correlate nicelywith the most mutable sites. 5-Methylcytosine hot spots in E. Nonsensemutations occurring at 15 different sites inlacI were scored. All result from theGC → AT transition. The asterisks (.) mark the positions of5-methylcytosines.How can 5-methylcytosines lead to mutations? One of the repair enzymes in thecell, - glycosylase, recognizes the uracil residues in the DNA thatarise from deaminations and excises them, leaving a gap that is subsequentlyfilled in. However, the deamination of 5-methylcytosine generates(5-methyluracil), which is not recognized by the uracil-DNAglycosylase and is thus not repaired.

Therefore, → transitions generatedby deamination are seen more frequently at 5-methylcytosine sites, becausethey escape this repair system.Oxidatively damaged bases constitute a third type of spontaneousimplicated in mutagenesis. Active oxygen species, such as superoxideradicals (O 2D), hydrogen peroxide (H 2O 2),and hydroxyl radicals (OHD), are produced as by-products of normal aerobic. These oxygen species can cause oxidative damage to, as wellas to precursors of DNA (such as GTP), resulting in.

Such mutationshave been implicated in a number of human diseases. Shows two products of oxidative damage.The 8-oxo-7-hydrodeoxyguanosine (8-oxodG, or “GO”) product frequentlymispairs with, resulting in a high level of → transversions.

Spontaneous mutations and human diseasessequence analysis has revealed the mutational lesions responsible for anumber of human hereditary diseases. Many are of the expected simplebase-substitution or or addition type. However, some are morecomplex but reminiscent of previously discussed bacterial mutations,allowing us to suggest mechanisms that cause these human disorders.number of these disorders are due to deletions or duplications of repeatedsequences. For example, mitochondrial encephalomyopathies are a group ofdisorders affecting the central nervous system or the muscles (for example,Kearns-Sayre syndrome).

They are characterized by dysfunction ofmitochondrial oxidative phosphorylation and by changes in mitochondrialstructure. These disorders have been shown to result from deletions of DNAsequences that lie between repeated sequences. Depicts one of these deletions.

Note howsimilar it is in form to the spontaneous E. Coli deletionsshown in. A secondexample is Fabry disease. This inborn error of results frommutations in the X-linked encoding the α-galactosidase A. Manyof these mutations are gene rearrangements, resulting from either deletionsor duplications between short direct repeats.

All these deletions occureither by a slipped mispairing mechanism, such as that pictured in, or bybetween the repeated sequences. Sequences of wild-type (WT) mitochondrial DNA and of deleted DNA(KS) from a patient with Kearns-Sayre/chronic externalopthalmoplegia plus syndrome. The 13-base boxed sequence isidentical in both WT and KS and serves as a breakpoint for theDNA deletion.common mechanism responsible for a number of genetic diseases is theexpansion of a three-base-pair repeat, as in the fragile X syndrome. For this reason, theyare termed trinucleotide repeat diseases. Fragile X syndrome isthe most common form of inherited mental retardation, occurring in close to1 of 1500 males and 1 of 2500 females. It is manifested cytologically by afragile site in the X that results in breaks. Fragile Xsyndrome results from changes in the number of a (CGG) n repeat in the coding sequence of the FMR-1.How does repeat number correlate with the disease?

Humans normallyshow a considerable in the number of CGG repeats in theFMR-1 gene, ranging from 6 to 54, with the mostfrequent containing 29 repeats. The variation in the number of CGGrepeats produces a corresponding variation in the number of arginineresidues (CGG is an arginine ) in the FMR-1- encodedprotein.

Sometimes, unaffected parents and grandparents give rise to severaloffspring with fragile X syndrome. Such unaffected ancestors in ahave been found to contain increased copy numbers of the repeat, rangingfrom 50 to 200. For this reason, these ancestors have been said to carrypremutations. The repeats in these premutation allelesare not sufficient to cause the disease phenotype, but they are much moreunstable (that is, readily expanded) than normal alleles, and so they leadto even greater expansion in their offspring. (In general, it appears thatthe more expanded the repeat number, the greater the instability.) Thepeople with the symptoms of the disease have enormous repeat numbers,ranging from 200 to 1300. Expansion of the CGG triplet in the FMR-1 geneseen in the fragile X syndrome.

Normal persons have from 6 to 54copies of the CGG repeat, whereas those from susceptiblefamilies display an increase (premutation) in the number ofrepeats: normally transmittingThe proposed mechanism for these repeats is a slipped mispairing duringsynthesis, just as shown previously for the lacIinvolving a one-step expansion of the four-base-pair sequence CTGG. However,the extraordinarily high frequency of at the trinucleotide repeatsin fragile X syndrome suggests that in human cells, after a threshold levelof about 50 repeats, the machinery cannot faithfully replicatethe correct sequence and large variations in repeat numbers result.Other diseases also have been associated with expansion of trinucleotiderepeats. There are several general themes to these diseases. The wild-typeincludes a repeated sequence within its protein-coding region, andcorrelates with this repeat region’s undergoing a considerableexpansion.

The severity of the disease correlates with the number of repeatcopies. Taken together, these observations suggest that the expanded repeatsare indeed parts of polypeptides and that the abnormal polypeptidescontaining large repeats of a single somehow contribute to thedisease state.X-linked spinal and bulbar muscular atrophy (known as Kennedydisease) results from the of a three-base-pairrepeat—in this case, a repeat of CAG. Kennedy disease, which ischaracterized by progressive muscle weakness and atrophy, results frommutations in the that codes for the androgen.

Normal personshave an average of 21 CAG repeats in this gene, whereas affected patientshave repeats ranging from 40 to 52.Myotonic dystrophy, the most common form of adult muscular dystrophy, is yetanother example of sequence expansion causing a human disease. Susceptiblefamilies display an increase in severity of the disease in successivegenerations; this increased severity is caused by the progressiveof a CTG at the 3′ end of a transcript. Normal peoplepossess, on average, five copies of the CTG repeat; mildly affected peoplehave approximately 50 copies; and severely affected people have more than1000 repeats of the CTG triplet. Additional examples ofare still appearing—for instance, which has recentlybeen added to the list. Biological Repair MechanismsLiving cells have evolved a series of enzymatic systems that repair damage ina variety of ways. The low rate is indicative of theefficiency of these repair systems. Failure of these systems can lead to ahigher mutation rate.

Number of human diseases can be attributed to defects inDNA repair, as we shall see later. Let’s first examine some of the characterizedrepair pathways and then consider how the cell integrates these systems into anoverall strategy for repair.We can divide repair pathways into several categories: prevention of errors,reversal of damage, and postreplication repair. Direct reversal of damageThe most straightforward way to repair a is to reverse it directly,thereby regenerating the normal base.

Reversal is not always possible,because some types of damage are essentially irreversible. In a few cases,however, lesions can be repaired in this way. One case is a mutagenicphotodimer caused by UV light (see ). The cyclobutane photodimer can be repaired by aphotolyase that has been found in bacteria and lowereukaryotes but not in humans. The binds to the photodimer and splitsit, in the presence of certain wavelengths of visible light, to generate theoriginal bases. Thisenzyme cannot operate in the dark, so other repair pathways are required toremove UV damage. Photolyase that reverses the 6-4 photoproducts has alsobeen detected in plants and Drosophila.

Repair of a UV-induced pyrimidine photodimer by aphotoreactivating enzyme, or photolyase. The enzyme recognizesthe photodimer (here, a thymine dimer) and binds to it. Whenlight is present, the photolyase uses its energy to split thedimer into theAlkyltransferases also are enzymes that directly reverselesions. They remove certain alkyl groups that have been added to theO-6 positions of by such mutagens as nitrosoguanidine andethyl methanesulfonate. The methyltransferase from E.

Colihas been well studied. This transfers the methyl group fromO-6-methylguanine to a cysteine residue on the protein.When this happens, the enzyme is inactivated, so this repair system can besaturated if the level of alkylation is high enough. Excinuclease incision patterns by E.

Coli (left)and human enzymes. The red points indicate the incision patternsof a lesion, in this case a thymine dimer, which is shown inorange. (Courtesy of J. Sancar,Science 266, 1974, 1954.)Certain lesions are too subtle to cause a distortion large enough to berecognized by the general excision-repair system and its counterparts inhigher cells. Thus, additional specific excision pathways arenecessary. Base- is carried out byglycosylases that cleave N-glycosidic(base–sugar) bonds, thereby liberating the altered bases and generatingapurinic or apyrimidinic sites (; see ). The initial step in this process isshown in.

The resultingsite is then repaired by an AP site-specific repairpathway. Action of DNA glycosylases. Glycosylase removes an altered baseand leaves an AP site.

The AP site is subsequently excised bythe AP endonucleases diagrammed in Figure 7-24. Copyright © 1983 by John Wiley.)Numerous glycosylases exist. One, -DNA glycosylase, removes uracilfrom DNA. Uracil residues, which result from the spontaneous deamination of, can lead toa → if unrepaired. It is possible that the natural pairingpartner of in DNA is (5-methyluracil) rather than uracil soas to allow the recognition and excision of these uracil residues. If uracilwere a normal constituent of DNA, such repair would not be possible.All cells have endonucleases that attack the sites left after the spontaneousloss of single or residues. The APendonucleases are vital to the cell, because, as noted earlier,spontaneous depurination is a relatively frequent event.

These enzymesintroduce chain breaks by cleaving the phosphodiester bonds at.This initiates an excision-repair process mediated by three furtherenzymes—an, I, and DNA. Repair of AP (apurinic or apyrimidinic) sites. AP endonucleasesrecognize AP sites and cut the phosphodiester bond. A stretch of DNAis removed by an exonuclease, and the resulting gap is filled in byDNA polymerase I and DNA ligase. Lewin,Owing to the efficiency of the AP repair pathway, it can be thefinal step of other repair pathways.

Thus, if damaged base pairs can beexcised, leaving an AP site, the AP endonucleases can complete therestoration to the. This is what happens in the glycosylaserepair pathway. Postreplication repairSome repair pathways are capable of recognizing errors even after hasalready undergone. One example, termed themismatch-repair system, can detect such mismatches.Mismatch-repair systems have to do at least three things: 1.Recognize mismatched base pairs. 2.Determine which base in the mismatch is the incorrect one. 3.Excise the incorrect base and carry out repair synthesis.The second property is the crucial one of such a system. Unless it is capableof discriminating between the correct and the incorrect bases, themismatchrepair system cannot determine which base to excise to prevent afrom arising.

If, for example, a mismatch occurs as aerror, how can the system determine whether G or T is incorrect?Both are normal bases in. But replication errors produce mismatches onthe newly synthesized strand, so it is the base on this strand that must berecognized and excised.To distinguish the old, strand from the newly synthesized strand,the mismatch-repair system, best characterized in bacteria, takes advantageof a delay in the of the following sequence, which normallyoccurs after. The methylating is methylase, which creates6-methyladenine on each strand. However, it takes the adenine methylaseseveral minutes to recognize and modify the newly synthesized GATCstretches. During that interval, the mismatch-repair system can operatebecause it can now distinguish the old strand from the new one by thepattern. Methylating the 6 position of adenine does not affectbase pairing, and it provides a convenient tag that can be detected by otherenzyme systems.

Showsthe during mismatch. Note that only the oldstrand is methylated at GATC sequences right after replication.

Once themismatched site has been identified, the mismatch-repair system corrects theerror. Model for mismatch repair in E. BecauseDNA is methylated by enzymatic reactions that recognize the A ina GATC sequence, directly after DNA replication the newlysynthesized strand will not be methylated.

The “hemimethylated”DNAThe E. Coli recA, one of the genes of the SOS bypasssystem , also takespart in postreplication repair. Here the system stalls at aUV photodimer or other blocking lesions and then restarts past the block,leaving a single-stranded gap. The RecA product takes part in, a processin which the gap is patched by DNA cut from the sister molecule. This process is noterror-prone. Strategy for repairWe can now assess the overall repair system strategy used by the cell. Themany different repair systems available to the cell are summarized in on page 220.

It would beconvenient if enzymes could be used to directly reverse each specific. However, sometimes that is not chemically possible, and not everypossible type of damage can be anticipated. Therefore, a generalexcision-repair system is used to remove any type of damaged base thatcauses a recognizable distortion in the. When lesions are toosubtle to cause such a distortion, specific excision systems, such asglycosylases, or removal systems are designed.

To eliminateerrors, a postreplication mismatch-repair system operates; finally,postreplication recombinational systems eliminate gaps across from blockinglesions that have escaped the other repair systems. Repair defects and human diseasesSeveral recessive human genetic diseases are known or suspected to be causedby defective genes in repair systems. These defects often lead to anincreased incidence of because, as part of the general increasedlevel of, more mutations are produced in genes that can cause acell to become cancerous (see Chapter15).One -prone disease, xeroderma pigmentosum (XP), resultsfrom a defect in any one of eight genes involved in.

People suffering from this disorder are extremely prone toUV-induced skin cancers as a result of exposure to sunlight and have frequentneurological abnormalities. The difference in UV photosensitivity betweennormal and XP cells is evident from the survival curves in.