How is dna repaired when nucleotides are mismatched

Even this, however, is not sufficient to prevent mismatch formation. It is rarely possible to determine the X-ray structure of a DNA duplex to a sufficiently high resolution to reveal the presence of hydrogen atoms, hence other techniques are used whenever there is ambiguity over the precise nature of the base pair.

Minor tautomer mismatches are almost perfect mimics of Watson-Crick base pairs in overall shape but they do not have the same hydrogen-bonding atoms in the major and minor grooves see Figure 2 and Figure 3. Some examples of minor tautomer mismatches are shown in Figure 2 and Figure 3.

Inosine I is an analogue of guanosine lacking an amino group on the 2-position of the purine ring. Inosine occurs in RNA, where it participates in base pairing with A, C, and U in codon-anticodon interactions, thus contributing to the degeneracy of the genetic code. Deoxyinosine occurs only rarely in DNA, where it arises by deamination of deoxyadenosine, and is potentially mutagenic. A number of DNA duplexes containing deoxyinosine have been analysed by X-ray crystallography in an attempt to explain its mutagenicity.

It seems unlikely, therefore, that the mutagenicity of inosine can be explained on purely structural grounds. Some deoxyinosine-containing mismatches have surprisingly high thermodynamic stability and this might be a key factor it is possible that some repair enzymes recognize mismatches by inserting an amino acid residue into the duplex and destroying base pairing; in such cases stable mismatches will be recognized less well than unstable ones.

Mismatches generally destabilize the DNA duplex and give local melting, or opening up, of the double helix, to promote base flipping ; and this is one mechanism by which DNA repair enzymes recognize mutagenic lesions.

Therefore, in this respect, deoxyinosine-containing mismatches are particularly difficult to recognize. Molecular biologists have used the special properties of inosine-containing mismatches when designing hybridization probes based on knowledge of protein sequences.

Deoxyinosine is included in oligonucleotides in positions where there is a sequence ambiguity owing to the degeneracy of the genetic code. The high thermal stability of inosine-containing mismatches ensures that the oligonucleotide hybridizes efficiently to the target nucleic acid. The stability of inosine-containing mismatches relative to their guanine counterparts is probably best explained by the destabilizing effect of the 2-amino group of guanine on guanine-containing mismatch base pairs.

However, in some guanine-containing mismatches e. This 2-amino group is absent in inosine, so no such destabilization is observed. UV melting is a method for measuring the melting temperature T m of a DNA duplex, an indicator of duplex stability. Each sample was analysed by UV melting and the T m was determined. The melting curves are shown in Figure 6.

The mismatch-containing duplexes were found to have significantly lower melting temperatures than the duplex without mismatches:. All mismatches are destabilized by a significant amount relative to it, with significantly lower melting temperatures.

The destabilizing effect of mismatch base pairs on DNA duplexes is illustrated clearly by this type of UV melting experiment. Mismatch base pairs are generally different in shape from Watson-Crick base pairs and are usually thermodynamically unstable.

The two properties shape and stability are related because "mis-shapen" base pairs are unlikely to form stable base-stacking interactions within the DNA double helix. In some cases, hydrogen bonding of the heteroatoms to the surrounding water molecules is also inhibited, thus further destabilizing the base pair.

It is likely that DNA repair enzymes utilize all of these factors in mismatch recognition. Apart from being the secret of life, DNA is a chemical molecule and, as with other molecules, the phosphates, sugars and heterocyclic bases of DNA are susceptible to modification by reagents including chemical carcinogens, ionizing radiation and ultraviolet light. Cytosine is susceptible to hydrolytic deamination to uracil Figure 7. The conversion of cytosine to uracil gives rise to transition mutations if left uncorrected.

O 6 -Methylguanine can be formed when DNA is exposed to alkylnitrosoureas, such as N -nitrosodimethylamine NDMA and N -methyl- N -nitrosourea the major product of the reaction is N 7 -methylguanine, which is not mutagenic, because the modification does not perturb base pairing; Figure 8. The presence of O 6 -methylguanine in DNA can be very damaging because methylation at the O 6 -position changes the hydrogen bonding properties of guanine, thereby inducing G to A transition mutations Figure 9.

Thus, the proofreading domain of DNA polymerases forces the modified guanine base to accept thymine as a partner instead of cytosine and the enzyme allows a lesion to pass by uncorrected.

Specific methyltransferase enzymes have evolved to demethylate methylguanine before mutations become incorporated into the genome. These bases exist predominately in the 8-keto form, and their contribution to mutagenesis is the subject of much interest.

Modification at the 8-position does not directly affect the ability of adenine and guanine to form Watson-Crick base pairs, but the presence of the bulky oxygen atom increases their tendency to adopt the syn conformation, thereby providing possibilities for base mispairing. In contrast to O8G, O8A 8-hydroxyadenine is not strongly mutagenic: the modified adenine base retains a strong preference for thymine as a partner.

The G syn O8A anti base pair Figure 12 is interesting because it appears to be held together by four three-centered hydrogen bonds sometimes called bifurcated hydrogen bonds; Figure This arrangement is stable because it allows the 2-amino group of guanine to fulfil its hydrogen bonding capacity by interacting with the oxygen atom of O8A as well as with a neighbouring water molecule not shown in Figure As mentioned previously, any form of base-pairing that prevents the guanine 2-amino group from fulfilling its hydrogen bonding potential, with either the opposing base or neighbouring water molecules, will tend to be unstable relative to the individual unpaired bases.

The reaction of purine bases with hydroxyl radicals can also result in the formation of the 5',8-cyclo-2'-deoxynucleosides 5',8-cyclo-2'-deoxyadenosine and 5',8-cyclo-2'-deoxyguanosine Figure Unlike 8-oxoadenine and 8-oxoguanine, the 5',8-cyclo-2'-deoxynucleosides cannot be repaired by base excision repair, and must be repaired by nucleotide excision repair. Replication errors can also involve insertions or deletions of nucleotide bases that occur during a process called strand slippage.

Sometimes, a newly synthesized strand loops out a bit, resulting in the addition of an extra nucleotide base Figure 3. Other times, the template strand loops out a bit, resulting in the omission, or deletion, of a nucleotide base in the newly synthesized, or primer , strand. Regions of DNA containing many copies of small repeated sequences are particularly prone to this type of error.

DNA polymerase enzymes are amazingly particular with respect to their choice of nucleotides during DNA synthesis, ensuring that the bases added to a growing strand are correctly paired with their complements on the template strand i. Nonetheless, these enzymes do make mistakes at a rate of about 1 per every , nucleotides. That might not seem like much, until you consider how much DNA a cell has.

In humans, with our 6 billion base pairs in each diploid cell, that would amount to about , mistakes every time a cell divides! Fortunately, cells have evolved highly sophisticated means of fixing most, but not all, of those mistakes. Some of the mistakes are corrected immediately during replication through a process known as proofreading , and some are corrected after replication in a process called mismatch repair. During proofreading, DNA polymerase enzymes recognize this and replace the incorrectly inserted nucleotide so that replication can continue.

After replication, mismatch repair reduces the final error rate even further. Incorrectly paired nucleotides cause deformities in the secondary structure of the final DNA molecule. During mismatch repair, enzymes recognize and fix these deformities by removing the incorrectly paired nucleotide and replacing it with the correct nucleotide.

Incorrectly paired nucleotides that still remain following mismatch repair become permanent mutations after the next cell division. This is because once such mistakes are established, the cell no longer recognizes them as errors. Consider the case of wobble-induced replication errors. When these mistakes are not corrected, the incorrectly sequenced DNA strand serves as a template for future replication events, causing all the base-pairings thereafter to be wrong.

For instance, in the lower half of Figure 2, the original strand had a C-G pair; then, during replication, cytosine C is incorrectly matched to adenine A because of wobble. In this example, wobble occurs because A has an extra hydrogen atom.

In the next round of cell division, the double strand with the C-A pairing would separate during replication, each strand serving as a template for synthesis of a new DNA molecule. At that particular spot, C would pair with G, forming a double helix with the same sequence as its original i.

This type of mutation is known as a base, or base-pair, substitution. Base substitutions involving replacement of one purine for another or one pyrimidine for another e. Likewise, when strand-slippage replication errors are not corrected, they become insertion and deletion mutations. Much of the early research on strand-slippage mutations was conducted by George Streisinger in the s. Streisinger, a professor at the University of Oregon and a fish hobbyist, is known by some as the "founding father of zebrafish research.

Streisinger used this virus to show that most nucleotide insertion and deletion mutations occur in areas of DNA that contain many repeated sequences also called tandem repeats , and he formulated the strand-slippage hypothesis to explain why this was the case Streisinger et al. In Figure 3, notice the series of repeat T's on the template strand where the slippage has occurred. When slippage takes place, the presence of nearby duplicate bases stabilizes the slippage so that replication can proceed.

During the next round of replication, when the two strands separate, the insertion or deletion on either the template or primer strand, respectively, will be perpetuated as a permanent mutation. Scientists have collected enough evidence to confirm Streisinger's strand-slippage hypothesis, and this type of mutagenesis remains an active field of scientific research. Figure 3: Strand slippage during DNA replication.

When strand slippage occurs during DNA replication, a DNA strand may loop out, resulting in the addition or deletion of a nucleotide on the newly-synthesized strand. Although most mutations are believed to be caused by replication errors, they can also be caused by various environmentally induced and spontaneous changes to DNA that occur prior to replication but are perpetuated in the same way as unfixed replication errors.

As with replication errors, most environmentally induced DNA damage is repaired, resulting in fewer than 1 out of every 1, chemically induced lesions actually becoming permanent mutations.

The same is true of so-called spontaneous mutations. Rather, they are usually caused by normal chemical reactions that go on in cells, such as hydrolysis. These types of errors include depurination , which occurs when the bond connecting a purine to its deoxyribose sugar is broken by a molecule of water, resulting in a purine-free nucleotide that can't act as a template during DNA replication, and deamination , which results in the loss of an amino group from a nucleotide, again by reaction with water.

Again, most of these spontaneous errors are corrected by DNA repair processes. But if this does not occur, a nucleotide that is added to the newly synthesized strand can become a permanent mutation. Mutation rates vary substantially among taxa, and even among different parts of the genome in a single organism. Scientists have reported mutation rates as low as 1 mistake per million 10 -8 to 1 billion 10 -9 nucleotides, mostly in bacteria , and as high as 1 mistake per 10 -2 to 1, 10 -3 nucleotides, the latter in a group of error-prone polymerase genes in humans Johnson et al.

Even mutation rates as low as 10 can accumulate quickly over time, particularly in rapidly reproducing organisms like bacteria.

This is one reason why antibiotic resistance is such an important public health problem; after all, mutations that accumulate in a population of bacteria provide ample genetic variation with which to adapt or respond to the natural selection pressures imposed by antibacterial drugs Smolinski et al. Take E. The genome of this common intestinal bacterium has about 4. Assuming a mutation rate of 10 -9 i.

That may not seem like much. At that point, approximately 10, of these bacteria will have accumulated at least one mutation.

As the number of bacteria carrying different mutations increases, so too does the likelihood that at least one of them will develop a drug-resistant phenotype. Likewise, in eukaryotes, cells accumulate mutations as they divide. In humans, if enough somatic mutations i. Or, less frequently, some cancer mutations are inherited from one or both parents; these are often referred to as germ-line mutations.

One of the first cancer-associated somatic mutations was discovered in , when researchers found that a mutated HRAS gene was associated with bladder cancer Reddy et al. HRAS encodes for a protein that helps regulate cell division. Since then, scientists have identified several hundred additional "cancer genes.

Of course, not all mutations are "bad. However, too much of a good thing can be dangerous. Induced mutations are those that result from an exposure to chemicals, UV rays, X-rays, or some other environmental agent. Spontaneous mutations occur without any exposure to any environmental agent; they are a result of natural reactions taking place within the body.

Mutations may have a wide range of effects. Some mutations are not expressed; these are known as silent mutations. Point mutations are those mutations that affect a single base pair. The most common nucleotide mutations are substitutions, in which one base is replaced by another. These can be of two types: transitions or transversions. Transition substitution refers to a purine or pyrimidine being replaced by a base of the same kind; for example, a purine such as adenine may be replaced by the purine guanine.

Transversion substitution refers to a purine being replaced by a pyrimidine or vice versa; for example, cytosine, a pyrimidine, is replaced by adenine, a purine. Mutations can also be the result of the addition of a base, known as an insertion, or the removal of a base, known as a deletion.

Sometimes a piece of DNA from one chromosome may get translocated to another chromosome or to another region of the same chromosome. Privacy Policy. Skip to main content. DNA Structure and Function. Search for:. DNA Repair. DNA Repair Most mistakes during replication are corrected by DNA polymerase during replication or by post-replication repair mechanisms.

Learning Objectives Explain how errors during replication are repaired.