LECTURE 9  Frameshifts, deletions and "dynamic" mutations


1. Frameshifts. 

            a. Frameshift mutation are caused by the addition or subtraction of nucleotides from the DNA sequence. Because the genetic code is read in triplets, addition or subtraction of 1 or 2 nucleotides causes a shift in the reading frame. Incorrect amino acids are inserted and often premature termination occurs when an nonsense codon is read. Frameshifts therefore cause, in general, very severe phenotypic effects.  Addition or subtraction of 3 nucleotides does not generally cause a mutation because the reading frame is not shifted. An extra amino acid is inserted or removed but this does not usually lead to perturbed protein structure. (This very fact lead to the deduction in the early 1960's that the genetic code is read in triplets). 

            b. Frameshifts do not occur at random places in a gene but "hot-spot" at runs of repeated sequences, especially AT-rich. 

            c. Streisinger et al. in 1966 proposed a "slippage" model whereby realignment of template and nascent strand during DNA replication, leading to a "looping out" of one of the two strands, causes frameshift. This model explains why frameshifts occur primarily at repeated runs of bases. 

            d. Certain compounds are very specific for the induction of frameshift mutation and do not lead to any increase in base substitution errors. These include: acridines, proflavin, ICR191, ethidium bromide. These agents all are multicyclic ring compounds and have some capacity to intercalate in DNA via base stacking interactions. However, the ease of intercalation is not correlated with the mutagenicity of these compounds -one idea is that these compounds stack and stabilize the swiveling DNA strands OUT of the helix in the slipped misalignment intermediate. 

            e. Frameshifts are a very common source of spontaneous mutations. In some genes they are the predominant type of mutation. As frameshifts are very context-sensitive (especially to repetitive sequences), the prevalence of frameshifts varies from locus to locus. Frameshifts are well-recognized by the MutSHL mismatch repair system and in mismatch repair-defective strains, frameshifts predominant the mutational spectrum. A defect in mismatch repair underlying HNPCC (hereditary nonpolypsis colon cancer) was deduced by the instability of minisatellite (short tandem repeat) arrays in cells derived from cancer patients. 

            f. Frameshift mutations are revertible by frameshift mutagens (i. e. +1 -1=0) but not by mutagens that induce base substitutions. Deletion mutations are nonrevertable by either treatment. By this criterion, early genetic experiments could therefore distinguish this type of mutation from base substitutions (such as those that lead to nonsense mutations) and deletions. 

2. Deletions.

            a. Deletions are the removal of a portion of a gene > a few nucleotides. (Otherwise they are considered - frameshifts.) Deletions can account for a substantial proportion of spontaneous mutations. They can occur over several nucleotides, short gene-size distances or include large portions of chromosomes. Deletion mutations encompassing specific chromosomal regions are often associated with progression of tumors.

            b. Like frameshift mutations, the prevalence of deletions is context-sensitive. Deletions are considered "illegitimate" recombination events because they were originally thought to occur at non-homologous sequences. However, sequence analysis of deletion mutations (Albertini et al 1982) shows that deletion mutations do not occur at random but tend to occur at short repeated sequences (5-20 bp in length) so limited homology may be important even for illegitimate recombination. Hot spots for deletion are such repeated structures associated with inverted repeat (possible hairpin structures).

            c. Some deletions occur between much larger repeats: between tandem arrays of duplicated genes (e. g. the globin families) or between dispersed repeats (e. g. IS  or REP elements in bacteria, retron or retrotransposon elements in eukaryotes). Several prevalent human genetic syndromes fall into this category: male color-blindness, thalessemias.

            d. Several mechanisms may account for deletions: recombination including unequal crossing-over, replication "slippage"  and annealing of broken and processed ds ends (“single-strand annealing”. Lovett et al. proposed that deletions can occur as a result of mispairing of sister-strands during recombinational repair of a blocked replication fork.  Most likely, each of these mechanisms can account for a subset of observed deletion mutations. 

3.  Duplications/expansion of tandem repeat arrays.  

            a. Like deletions, gene duplications or amplifications can arise at short or long repeated sequences.  Some gene families are found as clusters of tandem repeats that have diverged to various extents. This type of gene duplication is thought to have played a role in genomic evolution. Demand for increased expression of a gene sometimes may lead to gene amplification in tandem direct repeat arrays. Resistance to high levels of antibiotics in bacteria can result from this type of array. rRNA genes in many organisms are also present in tandem arrays. 

            b. Very short sequences are sometimes found in tandem arrays. Expansion of short (triplet) sequence repeats is associated with several human genetic diseases: myotonic muscular dystrophy, fragile-X syndrome, Kennedy's disease and Huntington's disease. The severity of the disease symptoms correlates with the size of the repeated array. The size of the repeated array tends to increase with subsequent generations. This explains the genetic phenomenon known as “anticipation” where the severity of the disease increases with each generation of passage. Such "mutations" are now termed "dynamic" mutations because the size of the array is unstable from generation to generation and also, in some cases, within different cells of an individual. 

            c. Unequal crossing-over, sister-strand exchange and replication "slippage" may account for these rearrangements. Special structures formed by such sequence repeats such as hairpins may account for their tendency to expand.

4. Repeated sequences and mismatch repair

a.  Tandem arrays can be stabilized by heterologies within the repeats.  Among people with trinucleotide repeat associated diseases, this reduces the severity of disease and the likelihood of inheritance.

b. Mismatch repair stabilizes short repeat arrays.  This as demonstrated in bacteria and in yeast.  This phenomenon explains the repeat instability phenotype observed in tumor cells from patients with HNPCC, hereditary nonpolyposis colon cancer.  Most HNPCC patients have an inherited mutant allele of hMSH2 ( a mutS homolog) or hMLH1 (a mutL homolog).  The second functional allele is lost by mutation or recombination (“LOH” or loss of heterozygosity).  This loss is frequent enough to make HNPCC appear to be caused by a genetically dominant mutation.

c. Mismatch repair also aborts recombination between larger homologies, if there are sequence differences (heterologies) between the repeated sequences.  This was first demonstrated for conjugational crosses between bacteria Salmonella and E. coli, which normally recombine very poorly (their genomes are about 88% identical on the DNA sequence level).  The level of interspecies recombination is elevated in all mismatch repair mutants (mutHLS dam etc.)  this type of recombination between diverged homologous sequences is called “homeologous” recombination.