Evolvability of SSRs and their Effect on Cellular Functions

Non-B DNA structures have been found to account for genomic instability in eukaryotes as well as prokaryotes (23,46). Among these different structural elements mutagenic effects on DNA have especially been associated to SSRs with high mutation frequency (177). Mutational effects include contractions (deletion) and expansions (insertion) of an SSR (Figure 11A) as well as translocations (Figure 11B). Although these changes in genomic material are primarily associated with deleterious effects for an organism and represent a predisposition for disease, they also empower an organism to undergo rapid evolutionary changes. SSR variability provides local sequence variation and thereby increases the organism’s fitness in a fluctuating environment (27,28,43,78,131,140,178,179). Changes as small as 1 nt are possible as well as rearrangements of several kilobases (28). Two mechanisms have been discussed that bring about variability of tandem repeats: recombination (Figure 11B and C) and strand slippage mispairing (SSM) (Figure 11D) (180). Repetitive sequences with large unit size are thought to be evolved by recombination rather than SSM and vice versa for short unit size (179,181-183). For SSRs with unit sizes of 1-3 nt it has been observed that the variability of repetitive sequences increases with the number of tandem repeats (184).

Figure 11: Mechanisms Leading to Repeat Expansion or Contraction

A: Contraction or expansion of the repetitive tracts leads to local sequence variability. B: Unequal crossing over occurs intermolecularly between two homologous repeat regions resulting in an expanded and a contracted repetitive sequence. Repeat units are represented as colored boxes. C: Intramolecular recombination leads to a deletion of a portion of the repeat sequence. D: After DNA polymerase (green) dissociation during replication strand slippage may occur. Strands may improperly realign at repetitive tracts. Depending on the site of false alignment being located in the nascent strand (red boxes) or in the template strand (blue boxes) expansion or contraction of the repetitive sequence will occur. Figures B-C are adapted from (141).

In E. coli orientation of a sequence with respect to the origin can influence mutability, furthermore G-tracts were shown to exhibit higher mutation rates than A-tracts (185). On the one hand increased microsatellite instability was observed in cells with impaired mismatch repair (185-187), which points towards a replicative mechanism. Mismatch repair may become saturated during fast growth conditions, DNA damage or inhibition e.g. by carcinogens, which will increase the propensity of SSRs to mutate (188). On the other hand double strand breaks may occur and recombination can be initiated during double strand break repair (189).

Unequal crossing over occurs between two DNA molecules carrying homologous repetitive elements as depicted in Figure 11B. The process yields two different recombination products: one with an expanded and one with a contracted repeat tract. In contrast intramolecular recombination as shown in Figure 11C takes place between repetitive sequences located on the same DNA strand, which will lead to a deletion within the repetitive tract. The classic example for sequence variability through recombination is the type IV pilin antigenic variation in N.

gonorrhoeae. Unidirectional transfer of genetic material occurs from one of the silent pilS loci to the expression locus pilE in a RecA dependent reaction (28,66,190).

Changes in the length of microsatellites are primarily thought to stem from replication slippage.

During replication the nascent DNA strand can denature from the template and realign. In case of repeats containing palindromic elements part of the repetitive sequence may loop out into hairpin or stem loop structures and while the rest of the single stranded repeat misaligns upon continuation of replication. This results in deletion or addition of repeat units depending on the looped-out sequence being present in the template strand or in the nascent strand, respectively (Figure 11D) (180,191).

By inducing local genetic instability SSRs have been shown to act as cis-regulatory motifs enabling the modulation of gene expression in a reversible manner, especially in phase and antigenic variation (27,28,180). In E. coli SSR mutability was found increased when cells were exposed to environmental stresses such as irradiation, elevated temperature or starvation conditions (192).

Both processes described above allow the switching of phenotypes in a bacterial population and thereby increase the organism’s fitness. Phase variation can be achieved by different mechanisms depending on the location of a repeat on the genome (Figure 12).

Variation of gene expression by SSRs located upstream of an ORF in the non-coding region can be achieved by repeats overlapping with regulator protein binding sites or changing the spacing between functional elements in promoters. For example, in N. meningitidis 5’-TAAA-3’ runs are present upstream of the -35 site in the core promoter of the nadA gene, which codes for NadA, an outer membrane protein and adhesin of the pathogenic bacterium. Variability in the number of repeats affects the binding of transcription factors, which results in three distinct transcription levels depending on the repeat copy number (193). Variation in the number of dinucleotide repeats present between the -35 and -10 site in the overlapping, but divergent, promoters of the

hifA and hifB genes in H. influenza affects the binding efficiency of the RNA polymerase and thereby transcriptional initiation, resulting in different expression levels of both genes (194). Phase variation by SSRs located within coding regions is often achieved by frame-shifting and production of aberrant or truncated protein variants due to an alternative stop codon. Any change in nucleotide number that is not a multiple of three will lead to the disruption of an ORF. The pentameric repeat 5’-CTCTT-3’ is found in the 5’ region of the opaC gene in N. gonorrhoeae encoding the opacity leader peptide. Whether translation occurs in or out of frame is dependent on the number of repeats present. While all expression loci of opacity proteins are constitutively transcribed, the production of functional proteins is determined on the translational level as only certain configurations of repeat numbers lead to coding regions that are still in frame with the start codon (ON state) whereas others lead to the production of aberrant or truncated gene products (OFF state) (27,195).

Figure 12: Genomic Locations of Repetitive Sequences Involved in Phase Variation

Phase variation in bacteria can be achieved by the local sequence variability of repeats located in coding and non-coding regions. Repeats in non-coding regions can have regulative effects when they are found within the promoter region or the 5’ UTR. ① Repeats located upstream of the core promoter in the so-called upstream activating sequence (UAS) can influence the binding of activators or repressors. ② Within the core promoter binding of the RNA polymerase can be affected by changing the spacing between the conserved -35 and -10 regions that are essential for polymerase binding.

③ Downsteam of the promoter spacing between the transcription start site (+1) and the ribosomal binding site (SD) can be influenced. ④ Variability of repeats within coding regions can lead to altered or truncated gene products.