Lecture 17: Point Mutations

Although the notes will cover most of what I discuss in class for this lecture, you should also read Chapter 6 to page 193. This will help fill in some of the details for parts that I only gloss over in these notes.

Chemical mutagenesis

Several types of chemical changes result in DNA damage. Depurination results from hydrolysis of the purine base A or G from the deoxyribose phosphate backbone. DNA replication of a depurinated strand will result in the insertion of a random base resulting in a mutation 3/4 of the time. Deamination is another alteration that results in mutation. Removal of the amino group from cytosine changes the base to uracil, a base found in RNA. U then pairs with A rather than G, resulting in a transition. Oxidation of guanine produces 8-oxoG, which pairs with A instead of C, resulting in a transversion.

Geneticists stimulate the formation of mutations with mutagenic agents. When the goal is to produce deletions or other rearrangements, X rays or gamma rays are used. When point mutations in a specific gene are desired, high energy radiation can be used, but chemical mutagens are usually chosen. These mutagens fall into several classes. Base analogs are similar in structure to a base and are incorporated during replication, but they have base pairing interactions from the normal base. 5-Bromouracil is almost identical to thymine and gets incorporated into DNA in place of thymine. Like thymine in usually pairs with A, but is an alternate isomeric state it pairs with G resulting in an A to G transition.

Several mutagens alter the structure of bases resulting in changes in base pairing. Hydroxylating agents add a hydroxyl to bases and result in pairing changes. Hydroxylamine adds a hydroxyl to C; hydroxylated C now pairs with A, resulting in a transition. Alkalating agents add methyl or ethyl groups to bases and change their pairing specificity. EMS adds an ethyl group to G or T, and modified G pairs with T and modified T pairs with G resulting in transitions. Deaminating agents remove amines to result in mispairing. Nitrous acid, for example, deaminates C changing it to U. It also deaminates A to hypoxanthine, a base that pairs with C.

The final class of chemical mutagens that we will discuss is intercalating agents. The mutagens are planar molecules that intercalate into DNA, resulting in a single base pair insertion or deletion.

What are the consequences of point mutations?

Point mutations can alter the protein of a single gene in several ways. Missense mutations result in single amino acid substitutions. Missense mutations may not affect protein function, or they may cause partial or complete loss of protein function. The effect of missense mutations on protein function depends on which amino acid is changed and the nature of the change. For example, some amino acids, often those on the surface, are relatively insensitive to changes, whereas other amino acids, for example, amino acids at the active site of the enzyme are very sensitive to changes. Some missense mutations are silent because they do not affect the function of the protein; others cause partial or complete inactivation of the proteins function.

Nonsense mutations change codons that specify a specific amino acid into a stop codon. This results in a truncated protein that often has no function. In addition, by a largely unknown mechanism, many nonsense mutations also cause the mRNA encoding the truncated protein to become unstable. Most chemically induced mutations are missense and nonsense mutations.

Frameshift mutations insert or delete one or two nucleotides and shift the reading frame. Splice site mutations change splice donor and acceptor sequences and can cause exons to be deleted or intron sequences to be included in the processed RNA. These changes can causes altered proteins to be synthesized

Missense mutations

The affect of missense mutations depends on the nature of the amino acid substitution and the position of the change in the protein. Some missense mutations do not interfere with protein function. Conservative amino acid substitutions replace an amino acid with another amino acid of similar chemical structure and may have no affect on protein function. Non-conservative amino acid substitutions caused by missense mutations are likely to disrupt protein function. For example, sickle cell hemoglobin is caused by a single missense mutation in the b-globin gene that changes a glutamate, which is positively charged, to a valine, which is an uncharged hydrophobic amino acid.

The effect of an amino acid substitution on a protein depends on the role of the particular residue in protein activity. Using in vitro mutagenesis, it is possible to test which amino acids are necessary for protein function. For example, the HIV-1 protease is the enzyme that recognizes one of the proteins encoded by HIV-1, the retrovirus that causes AIDS. Because the protease is small, only 99 amino acids, it is possible to systematically study each amino acids role in protease function. 330 different mutations were made alter each amino acid in at least one way. The mutants were then assayed for enzyme activity. Non-conservative substitutions in 39 of 99 amino acids destroyed enzyme activity. Even though this is a small protein only 40% of the amino acids are essential for function. Those amino acids not affected by changes usually lie on the surface of the protein.

Nonsense and frameshift mutations

Nonsense and frameshift mutations produce truncated protein products. Nonsense mutations are single nucleotide substitutions that change a codon that encodes an amino acid to one of three stop codons: TGA, TAG or TAA. If the nonsense codon is near the 5' end of the open reading frame that encodes the amino terminus of the protein the truncated protein that is produced will lack any function. Nonsense mutations near the carboxyl terminus can produce truncated proteins with partial or complete function. By unknown mechanisms, nonsense mutations often result in unstable mRNAs.

Frameshift mutations are insertions or deletions of one or two nucleotides in the coding region of a gene. As a consequence of the insertion or deletion, the reading frame is shifted after the site of the deletion. Because 3 out of the 64 codons are stop codons, translation though the new out of frame mRNA will encounter a stop codon within approximately 20 amino acids, on average.

Mutations that disrupt splicing and transcription

Mutations that interfere with the function of hemoglobin have identified many of the amino acids important in hemoglobin function. Mutations that interfere with the function of hemoglobin, the oxygen carrying protein in red blood cells, are identified as genetic diseases that cause anemia. Hemoglobin in adults is a tetramer of the types of subunits: each hemoblobin contains two a subunits and two ß subunits. Humans have two genes that encode the a subunit, but only one gene that encodes the ß subunit. As a result points mutations in the in the ß gene may cause anemia, but mutations in one a gene does not. The mutations either produce altered protein products or reduce the levels of one of the two globins. Lowering hemoglobin levels causes an anemia called thalassemia.

a-thalassemias blocks the production of a-globin, and ß-thalassemias block the production of ß-globin. Because there are two a genes, a-thalassemias are usually caused by a deletion that removes the adjacent a genes. By contrast most ß-thalassemias are caused by point mutations. A large fraction of these mutations prevent normal splicing of the primary transcript. Some mutations that block normal splicing map to splice donor sequences in introns, changing the invariant GT to AT, TT or GG. Other mutations change the invariant AG in the splice acceptor sequence. These splice donor and acceptor mutations all block the production of any functional ß-globin protein. Other mutations within the ß-globin introns create new splice donor or acceptor sequences.

Rare point mutations outside coding regions cause mutant phenotypes. There are many elements in non-coding DNA with important functions in transcription, and mutations in these elements can cause defects. For example, some ß-thalassemias are caused by mutations in the 5' promoter of the ß-globin gene.

Mutations in bacterial operons can affect several genes.

Nonsense or frameshift mutations in bacterial operons can block the synthesis of several proteins. Operons are transcribed from a single promoter into a single mRNA that encodes all of the proteins of the operon. These mRNAs are called polycistronic mRNAs because more than one gene (cistron) is encoded in the mRNA. Upon completion of translation of the first protein, translation of the second protein begins without the ribosome ever leaving the mRNA. A nonsense mutation in the first coding region of the polycistronic mRNA will cause the ribosome to dissociate from the mRNA before the second reading frame is translated. Because there are no signals in the mRNA that allow ribosomes to begin translation in the meddle of the gene, no proteins 3' to the nonsense mutation are translated. Thus nonsense mutations in prokaryotes have polar effect on downstream genes.