A single base mutation occurs during replication of a genome

a) Describe how that mutation may have occurred during replication

A point mutation describes the occurrence of a single base in a genome being changed for another (mismatch), removed from the replicated genome (deletion) or an extra base being incorporated incorrectly in its place (insertion). Substitution mutations can be either silent, missense or nonsense. In a silent mutation, the base is substituted for a base that encodes for a synonymous codon, resulting in an unchanged amino acid sequence upon translation. A missense mutation results in the generation of a codon that encodes for a different amino acid than the template strand would have produced. A nonsense mutation results in an incorrectly placed stop codon, which truncates translation. Both missense and nonsense mutations result in an incorrect, and likely dysfunctional, polypeptide structure, and can be caused by a number of various errors in DNA replication. A base change that results in a purine base from a purine base is known as a transition mutation.

The result of a purine to pyrimidine mutation is known as a transversion mutation. Tautomeric shifts are one of the most prominent errors observed in DNA replication and result in base-pair mismatching. All bases in DNA exist as tautomers - different chemical forms of the molecule in which protons are in different positions - but tend to exist in their more common 'keto' form. When a base shifts into its rarer 'imino' or 'enol' form, it may form hydrogen bonds with another base more entropically favourable than its common base-pair interaction. Another way bases can be mismatched is by simply shifting their position in space, resulting in an inappropriate bond forming between bases that are not commonly paired (e.g. thymine-guanine), in a process known as wobbling. This tends to occur with bases uracil and guanine, as the pairing is less specific. Replication errors involving insertion or deletion occur due to strand slippage. This is when, during replication, the template or newly synthesised strand loops slightly out of the replication mechanism, bypassing the replication machinery, resulting in the omission, if the parent template strand loops, or addition, if the daughter strand loops, of a nucleotide base. Sometimes mutations occur during replication due to interactions of DNA with the environment. Ultraviolet (UV) light can be detrimental to DNA in skin cells by causing a substitution of a cytosine base in the template strand to a thymine nucleotide in the newly synthesised strand. This occurs through deamination, where the hydrolysis of cytosine, turning it into uracil, causes the base to mispair with adenine during replication, and ultimately be replaced by thymine. Oxidising agents also interact with DNA from the environment by altering the base-pairing ability of nucleotides with chemical modifications. Benzopyrene, a known carcinogen, has been shown to produce lesions at guanine bases in some genes, causing problems in the base's ability to pair to cytosine. Depurination is another process that results in a single base mutation during replication. In this process, a purine base is lost through hydrolysis, but the sugar-phosphate backbone unaltered, spontaneously without environmental input. Without repair, this can result in the incorporation of an incorrect base in the next round of replication.

b) How might that mutation be recognised and repaired?

In order to tackle mutations caused by mis-incorporated, inserted or deleted bases, cells exhibit the use of a system known as DNA mismatch repair. This first requires the distinction of the parent template strand from the newly synthesised daughter strand, as the process is strand-specific. Once this is achieved, a number of mismatch repair proteins can act on the strand to excise the incorrectly incorporated base. One of these proteins, MutS, first forms a dimer and binds the mutated DNA in the daughter strand after recognition of a mutated base. A complex is then formed between the protein dimerDNA complex and MutH, bound in the strand at hemimethylated sites. DNA is then looped out in search for the nearest methylation site to the mismatched base. Upon activation of the complex by contact from an MutL dimer, acting as a mediator between the MutS dimer and MutH, the daughter strand is nicked near the methylation site. DNA Helicase II is then recruited to separate the two strands from each other. Formation of a larger complex of repair enzymes then follows, which slides along the DNA until it reaches the mismatched base, liberating the strand to be excised as it moves. Exonuclease trails this complex and digests the tail of DNA. This process produces a site of excised DNA which can then be repaired by DNA polymerase III and a single-stranded-binding protein, sealed by DNA ligase, and methylated by a DNA methyltransferase. This process will reverse the detrimental effect potentially caused by mismatched bases during the replication process. If the mutated base in question was mutated through deamination or environmental damage, a base excision repair may also be used to repair the DNA. This mechanism makes use of DNA glycosylases, which recognise the damaged bases and remove them from the daughter strand after DNA replication. This forms an AP site, which is cleaved by an AP endonuclease. The resulting break is then processed by the addition of a single, correct nucleotide by DNA Polymerase I, and sealed by DNA ligase. This process of DNA repair acts on deaminated bases such as uracil and hypoxanthine. Some mistakes in DNA replication are recognised immediately, during replication, in a process known as proofreading. In prokaryotes, all DNA polymerases have a proofreading domain that utilises 3'—>5' exonuclease activity during excision of an incorrect base pair. Upon recognition of the incorrect base pair, DNA polymerase reverses its direction by a single base pair and removes the mismatched base, re-inserts the correct base and continues replication. In eukaryotes, only the proteins involved in elongation have proofreading ability, but work with a similar mechanism. Post-transcriptional proofreading also occurs in mRNA translation during protein synthesis, in which incorrect aminoacyl-tRNAs are removed from the amino acid sequence before peptide bonds are formed.

c) What if the mutation is in a eukaryotic genome and is not repaired and then disrupts splicing? Describe the potential consequences for expression of a gene.

The process of splicing describes the removal of introns from pre-mRNA complexes, leading to the mature mRNA that can then be translocated and translated into amino acid sequences. Mutations in splicing regions, and those that interfere with the cells ability to excise introns, can result in monumental polypeptide dysfunction related to a plethora of inherited diseases, . Introns are small regions of DNA that are present in precursor mRNA post-transcription, and are removed from the molecule before it is used as a template for an amino acid sequence (they are not expressed in the gene). They are non-coding, meaning they are not required for the translation outcome of a functional protein, and will most likely cause dysfunction if present in mature mRNA. Splice site mutations describe the occurrence of insertion, deletion or substitution of nucleotide bases in the site of premRNA at which splicing takes place. For example, mutations in the COL7A1 gene occur mostly within the "GT" at the 5' end or the 'AG' at the 3' end of introns. This causes exon skipping, where exons are shortened in part or simply cut out of the genome and are therefore not expressed. Mutation in a splice site can also disrupt the entire reading frame. If this disruption is in the form of a frameshift, almost every triplet codon after that point in the genome will encode the incorrect amino acid in the translated polypeptide. Premature exposure of a stop codon as a result of the loss of function of a splice site can end the transcription and/or splicing of the genome. The splicing process is also driven by a number of sequences, either splice-donor or splice-acceptor, which can be mutated themselves. This can result in the incorrect recognition of introns as exons, or vice versa, ultimately leading to the retention of intronic DNA or the excision of entire exons vital to the splicing machinery or the genome currently being encoded. In Childhood Absence Epilepsy, a point mutation in the splice-donor site of the GABRG2 gene is thought to produce a dysfunctional protein, potentially serving as the cause for the disorder. Overall, the process of splicing is open to a number of different mutations that, upon gene expression, can result in the expression of a gene that is almost entirely incorrect and will code for a dysfunctional protein. The expression of introns can also cause the production of proteins that are not needed and potentially disruptive to the required protein encoded for by the genome. These tend to include proteins that are used for the regulation of gene expression, synthetic machinery and other regulatory proteins. However, not all splicing mutations are detrimental to the function of the endpoint protein. Nonsense mutations in the splice site of some genes result in truncated proteins and accelerated mRNA decay of the mutant template strands. The degree of truncation of the exon in the encoded gene determines the effect the mutation has on the function of the protein. If an exon is shortened by a small number of base-pairs, there is a chance the removed bases and the codons they form, encode amino acids insignificant to the quaternary structure of the target genome.

d) What if the unrepaired mutation is in the coding region of a eukaryotic gene and introduces (1) a rare codon? Again, describe the potential consequences for expression of a gene.

Introduction of rare codons in target genomes can decrease the efficiency of translation of the mRNA and the expression of certain target proteins. Rare codons are the small coding triplets of genetic code which encode an amino acid, that are used on a much smaller scale than others in transcription. Other triplets are likely to encode the same amino acid, but their synonymous counterparts are not used with equal frequency. In expression of the gene, it is presumably unlikely that the introduction of a rare codon would cause many problems in the function of the final polypeptide. This is because of the degeneracy of the genetic code, in the sense that most rare codons encode for the same amino acid as the codon they are replacing. However, in eukaryotes it is possible for rare codons to simply arrest translation due to the prospect of low numbers of their cognate tRNAs. In doing so, the introduction of a rare codon would result in low levels of protein production and gene expression, as the proteins the gene encodes are likely to be short in length, dysfunctional, and/or not formed due to the lack of peptide bonds between amino acids localised to the template strand by tRNAs. However, the potential for the introduction of rare codons into coding regions of eukaryotic genes could also pose the potential for the utilisation of rare codons in the regulation of gene and protein expression. Codon bias, the biased frequency of synonymous codons, is generally widely observed in eukaryotes at the genome level, and provides an arguably important contribution to translation efficiency. This is achieved by fine-tuning the rate of the elongation step of the process. The efficiency of translation is partly determined by elongation rates, which is assumed to occur faster and/or of higher efficiency when involving the use of more frequently used codons. This is because they are recognised faster by their cognate tRNAs, the abundance of which is likely much higher than the synonymous rare codon counterparts, as a result of the higher requirement for the common codons than rare codons. This is most likely used in some physiological processes involving translation of DNA into protein, where the regulation of translation is beneficial to the efficiency of the process in question. In a mutated eukaryotic genome, this method of regulation by protein synthesis machinery is likely disrupted by the introduction, or lack thereof, of a rare codon. If a process relies on the occasional presence of a rare codon to slow the rate of elongation, yet a mutation causes a rare codon to be inserted prematurely or at random in the genome, then the possibility for the detrimental effect of overproduction of the protein expressed is created.