Frameshift mutation
A frameshift mutation is a genetic mutation caused by indels of a number of nucleotides in a DNA sequence that is not divisible by three. Due to the triplet nature of gene expression by codons, the insertion or deletion can change the reading frame, resulting in a completely different translation from the original. The earlier in the sequence the deletion or insertion occurs, the more altered the protein. A frameshift mutation is not the same as a single-nucleotide polymorphism in which a nucleotide is replaced, rather than inserted or deleted. A frameshift mutation will in general cause the reading of the codons after the mutation to code for different amino acids. The frameshift mutation will also alter the first stop codon encountered in the sequence. The polypeptide being created could be abnormally short or abnormally long, and will most likely not be functional.
Frameshift mutations are apparent in severe genetic diseases such as Tay–Sachs disease; they increase susceptibility to certain cancers and classes of familial hypercholesterolaemia; in 1997, a frameshift mutation was linked to resistance to infection by the HIV retrovirus. Frameshift mutations have been proposed as a source of biological novelty, as with the alleged creation of nylonase, however, this interpretation is controversial. A study by Negoro et al. found that a frameshift mutation was unlikely to have been the cause and that rather a two amino acid substitution in the active site of an ancestral esterase resulted in nylonase.
Background
The information contained in DNA determines protein function in the cells of all organisms. Transcription and translation allow this information to be communicated into making proteins. However, an error in reading this communication can cause protein function to be incorrect and eventually cause disease even as the cell incorporates a variety of corrective measures.Genetic information is conveyed by DNA for protein synthesis within cells. Misinterpretation can lead to faulty function and disease, despite cellular correction mechanisms.Central dogma
In 1956 Francis Crick described the flow of genetic information from DNA to a specific amino acid arrangement for making a protein as the central dogma. For a cell to properly function, proteins are required to be produced accurately for structural and for catalytic activities. An incorrectly made protein can have detrimental effects on cell viability and in most cases cause the higher organism to become unhealthy by abnormal cellular functions. To ensure that the genome successfully passes the information on, proofreading mechanisms such as exonucleases and mismatch repair systems are incorporated in DNA replication.Transcription and translation
After DNA replication, the reading of a selected section of genetic information is accomplished by transcription.Nucleotides containing the genetic information are now on a single strand messenger template called mRNA. The mRNA is incorporated with a subunit of the ribosome and interacts with an rRNA. The genetic information carried in the codons of the mRNA are now read by anticodons of the tRNA. As each codon is read, amino acids are being joined until a stop codon is reached. At this point the polypeptide has been synthesised and is released. For every 1000 amino acid incorporated into the protein, no more than one is incorrect. This fidelity of codon recognition, maintaining the importance of the proper reading frame, is accomplished by proper base pairing at the ribosome A site, GTP hydrolysis activity of EF-Tu a form of kinetic stability, and a proofreading mechanism as EF-Tu is released.
Frameshifting may also occur during prophase translation, producing different proteins from overlapping open reading frames, such as the gag-pol-env retroviral proteins. This is fairly common in viruses and also occurs in bacteria and yeast. Reverse transcriptase, as opposed to RNA Polymerase II, is thought to be a stronger cause of the occurrence of frameshift mutations. In experiments only 3–13% of all frameshift mutations occurred because of RNA Polymerase II. In prokaryotes the error rate inducing frameshift mutations is only somewhere in the range of.0001 and.00001.
There are several biological processes that help to prevent frameshift mutations. Reverse mutations occur which change the mutated sequence back to the original wild type sequence. Another possibility for mutation correction is the use of a suppressor mutation. This offsets the effect of the original mutation by creating a secondary mutation, shifting the sequence to allow for the correct amino acids to be read. Guide RNA can also be used to insert or delete Uridine into the mRNA after transcription, this allows for the correct reading frame.
Codon-triplet importance
A codon is a set of three nucleotides, a triplet that codes for a certain amino acid. The first codon establishes the reading frame, whereby a new codon begins. A protein's amino acid backbone sequence is defined by contiguous triplets. Codons are key to translation of genetic information for the synthesis of proteins. The reading frame is set when translating the mRNA begins and is maintained as it reads one triplet to the next. The reading of the genetic code is subject to three rules the monitor codons in mRNA. First, codons are read in a 5' to 3' direction. Second, codons are nonoverlapping and the message has no gaps. The last rule, as stated above, that the message is translated in a fixed reading frame.Mechanism
Frameshift mutations can occur randomly or be caused by an external stimulus. The detection of frameshift mutations can occur via several different methods. Frameshifts are just one type of mutation that can lead to incomplete or incorrect proteins, but they account for a significant percentage of errors in DNA. In an unaltered gene, codons are sequentially interpreted, with each codon encoding a specific amino acid. This is known as the standard reading frame. However, in cases of frameshift mutations, an extra nucleotide is inserted into the DNA sequence, disrupting the typical reading frame and causing a shift in the sequence.This insertion prompts a shift in the reading frame due to the triplet nature of the genetic code. For instance, the addition of an extra "A" leads to a sequence shift, triggering the reading of an entirely different set of codons. This deviation in genetic information causes the ribosome, which reads the mRNA for protein synthesis, to misinterpret the genetic data. Consequently, an entirely different series of amino acids is generated, resulting in the generation of an altered protein sequence. In most instances, the new reading frame results in an early encounter with a stop codon, leading to the formation of a shortened and usually inactive protein. This form of mutation is termed an early stop codon or a nonsense mutation.
Genetic or environmental
This is a genetic mutation at the level of nucleotide bases. Why and how frameshift mutations occur are continually being sought after. An environmental study, specifically the production of UV-induced frameshift mutations by DNA polymerases deficient in 3′ → 5′ exonuclease activity was done. The normal sequence 5′ GTC GTT TTA CAA 3′ was changed to GTC GTT T TTA CAA of GTC GTT C TTA CAA to study frameshifts. E. coli pol I Kf and T7 DNA polymerase mutant enzymes devoid of 3′ → 5′ exonuclease activity produce UV-induced revertants at higher frequency than did their exonuclease proficient counterparts. The data indicates that loss of proofreading activity increases the frequency of UV-induced frameshifts.Detection
Fluorescence
The effects of neighboring bases and secondary structure to detect the frequency of frameshift mutations has been investigated in depth using fluorescence. Fluorescently tagged DNA, by means of base analogues, permits one to study the local changes of a DNA sequence. Studies on the effects of the length of the primer strand reveal that an equilibrium mixture of four hybridization conformations was observed when template bases looped-out as a bulge, i.e. a structure flanked on both sides by duplex DNA. In contrast, a double-loop structure with an unusual unstacked DNA conformation at its downstream edge was observed when the extruded bases were positioned at the primer–template junction, showing that misalignments can be modified by neighboring DNA secondary structure.Sequencing
and pyrosequencing are two methods that have been used to detect frameshift mutations, however, it is likely that data generated will not be of the highest quality. Even still, 1.96 million indels have been identified through Sanger sequencing that do not overlap with other databases. When a frameshift mutation is observed it is compared against the Human Genome Mutation Database to determine if the mutation has a damaging effect. This is done by looking at four features. First, the ratio between the affected and conserved DNA, second the location of the mutation relative to the transcript, third the ratio of conserved and affected amino acids and finally the distance of the indel to the end of the exon.Massively Parallel Sequencing is a newer method that can be used to detect mutations. Using this method, up to 17 gigabases can be sequenced at once, as opposed to limited ranges for Sanger sequencing of only about 1 kilobase. Several technologies are available to perform this test and it is being looked at to be used in clinical applications. When testing for different carcinomas, current methods only allow for looking at one gene at a time. Massively Parallel Sequencing can test for a variety of cancer causing mutations at once as opposed to several specific tests. An experiment to determine the accuracy of this newer sequencing method tested for 21 genes and had no false positive calls for frameshift mutations.