DNA INVERTED REPEATS AND HUMAN DISEASE

John J. Bissler

The Children's Hospital Research Foundation, Cincinnati, Ohio

6. BIOLOGICAL ROLES OF INVERTED REPEATS

6.1. RNA Effects

There is clear evidence that inverted repeats affect transcription. Virtually every molecular biology textbook has a discussion about rho independent termination (24- 27). In this mechanism the inverted repeat from the 3' end of the mRNA facilitates termination of transcription. Furthermore, both ferritin and the transferrin receptor employ a iron response element (IRE) that regulates translation as a function of alternative secondary structure involving the IRE and polyribosome formation (28- 30). The IRE in ferritin mRNA is 28 nucleotides long at the 5' end; whereas, in transferrin receptor mRNA the IRE is in the 3' untranslated region. When iron is in low abundance, the ferritin mRNA does not form polyribosomes whereas the transferrin receptor mRNA is translated. When iron is adequate, ferritin mRNA forms polyribosomes and transferrin receptor mRNA is degraded. This likely is regulated by protein binding competing with DNA secondary structure formation.

Likewise, transcription regulation of the human enkephalin gene is dependent on a 23 bp imperfect palindrome, or quasipalindrome, sequence (nucleotides -84 to -106). This sequence appears to be crucial for gene regulation (31, 32). Finally, mutations in the type XVII collagen gene result in the disease bullous pemphigoid (33- 35). This gene also has an imperfect inverted repeat in the 5' end that has been postulated to regulate protein production (36).

Besides regulating protein production through mRNA intermediates, perfect and imperfect inverted repeats have structural roles in tRNA, ribozymes, and some ribonuclear proteins.

6.2. DNA effects

Inverted repeats have a number of biological roles. The easiest to understand is the DNA binding site for homodimeric proteins. Through interaction at their dimerization domains, the proteins become oriented in an antiparallel fashion. Their DNA binding domains also would be positioned in opposite directions, requiring an inverted repeat for binding. The classic example of this resides in restriction endonucleases. There is no evidence that these small inverted repeats extrude into secondary structure to achieve DNA cleavage.

In some prokaryotic systems, inverted repeats appear to be crucial for DNA replication. Escherichia coli contains a 245 bp origin of replication that contains many inverted repeats. However, there is no evidence that these inverted repeats function in an alternative conformation (37). Other systems actually require the inverted repeat to engage in intrastrand base pairing for function. The single-stranded bacteriophage G4 contains inverted repeats ranging from 20 to 44 base pairs long that are required for proper function of the origin of replication (38). The protein DnaG binds to such an inverted repeat and this interaction is required for initiation of replication. The plasmid pT181 contains an inverted repeat that actually functions as an extruded cruciform structure to initiate replication (39,38). The bacteriophage N4 employs an extruded cruciform, that binds SSB on the single-strand loop to promote transcription of early genes (40, 41).

Eukaryotic DNA replication systems can also employ inverted repeats. The SV40 virus contains inverted repeats that are critical for DNA replication; however, evidence thus far suggests their function is the linear form (42). The human pathogen HSV likewise employs inverted repeats. The genome contains inverted repeats oriL₁, a 144 bp inverted repeat and oriL₂ a 136 bp inverted repeat that is unstable when cloned into a bacterial system (43, 44). These examples raise the possibility that one strategy for replicating DNA employs inverted repeats, both in the duplex form with the complementary strand as well as in the extruded conformation.

Inverted repeats whether perfect or imperfect are imbedded in genomes and have critical functions. Yet, these structures pose a special impediment to DNA replication fidelity and are associated with several human disease related genes.