Mismatch Repair


Animation: Eukaryotic mismatch repair.


Mismatch repair (MMR) corrects mismatched nucleotides that arise from replication errors, suppresses recombination between non-allelic partially homologous sequences during DSB repair (h omeologous recombination, HEOR), and functions as a lesion sensor in the DNA damage response (DDR).


MMR is an excision-resynthesis reaction. The excision tract begins at a distant strand scission (nick) and proceeds to just past a mismatch or lesion. MutS homologs (MSH) and MutL homologs (MLH/PMS) are the most highly conserved components of MMR. Mismatch and lesion recognition requires a dimer (prokaryotes) or heterodimer (eukaryotes) of MSH protein. In human cells, base-base and small insertion/deletion loop-type (IDL) mispairs are detected by hMSH2-hMSH6 while large IDL mispairs are detected by hMSH2-hMSH3. The MLH/PMS proteins also function as dimers (prokaryotes) or heterodimers (eukaryotes) downstream of MSH recognition; although their exact function in MMR is largely unknown. The major human MLH/PMS heterodimer is hMLH1-hPMS2, although hMLH1-hMLH3 may partially substitute some functions. Eukaryotic MMR must contain a single-stranded DNA (ssDNA) scission 5’ or 3’ of the mismatch to initiate strand excision and the excision nuclease activity appears to be accomplished by a combination of Exonuclease I (ExoI; 5’-exonuclease) as well as an intrinsic ssDNA endonuclease encoded in some MLH/PMS family members. The minimal human 5’→3’ and 3’→5’ excision reaction requires hMSH2-hMSH6 (or hMSH2-hMSH3), hMLH1-hPMS2, ExoI, ssDNA binding protein Replication Protein A (RPA), Proliferating Cell Nuclear Antigen (PCNA), and Replication Factor C (RFC; Resynthesis of the excision gap appears to utilize the major replication polymerases δ and/or ε along with ligase I, and is enhanced by HMGB1. The detailed biophysical mechanism of MMR is largely unknown in any organism.


The Fishel laboratory has developed the Molecular Switch model that accounts for the known MMR component functions (see: eukaryotic MMR movie). Current studies use a combination of fundamental biochemistry, biophysics and single molecule imaging to examine the Molecular Switch Model and test critical predictions.




1. How is the MSH mismatch binding signal accurately transmitted to the DNA strand scission where MMR is initiated?

There is general agreement that MSHs bind mismatched nucleotides and DNA lesions and that MLH/PMS transmit that recognition signal to downstream events. In eukaryotes, the DNA strand scission that is required to initiate MMR may be located several hundred to several thousand nucleotides away from the mismatch. Single molecule imaging approaches have provided a visual confirmation for ATP-bound MSH sliding clamps. As an example, the use of single molecule flow förster resonance energy transfer energy (smFlow-FRET) is diagramed in Figure 1A and B. The system attaches a recombinant λ DNA containing a single mismatch to the surface of a custom flow cell via biotin-streptavidin linkage. The open end is blocked via digoxigenen-antidigoxigenin binding. The DNA is then stretched with a defined and controlled laminar flow. Binding of a Cy3-labeled MSH to the mismatch will result in FRET emission with a Cy5 dye that is located 9 bp 5’ of the mismatch (Figure 1C). When the MSH binds ATP and forms a sliding clamp, the FRET signal will disappear and only emission of the Cy3 will be visualized (Figure 1H).


Figure: Single-Molecule Tracking of MutS on DNA.

(A) Illustration of a 15.3 kb λ-based DNA used for smFlow-FRET.
(B) A schematic representation of smFlow-FRET using prism-type total internal reflection fluorescence (TIRF) microscopy.
(C) A representative kymograph that shows searching MutS (strong green signal), followed by mismatch binding (reduced green signal; increased red FRET) in the absence of ATP.
(D) Representative time trace of donor-acceptor intensity (top) and the resulting FRET efficiency (bottom).
(E) A histogram of the FRET efficiency obtained from MutS molecules (Emismatch = 0.71 ± 0.04; n = 46).
(F) A histogram of the dwell time for MutS at the unpaired dT in the absence of ATP (τmismatch = 32.2 ± 4.9 s; n = 48).
(G) The displacement of Cy3-MutS while it diffuses along the DNA (left, green) or bound to the mismatch exhibiting FRET indicated by Alexa647 emission (left, red). The mean square displacements (MSD) of Cy3-MutS (right, green) and Alexa647 FRET (right, red) were plotted versus time where the slope provides the diffusion coefficients of searching MutS (DMutS•searching = 0.035 ± 0.005 μm2 s-, mean ± s.e.) and mismatch bound MutS (DMutS•mismatch = 0.002 ± 0.002 μm2 s-1, mean ± s.e.) in 50 mM NaCl.
(H) A representative kymograph of Cy3-MutS mismatch interaction(s) in the presence of ATP (200 μM). FRET emission by Alexa647 (red) indicates mismatch binding, followed by the formation of an ATP-bound MutS sliding clamp.
(I) Representative time trace of a donor Cy3-MutS and acceptor Alexa647 intensity (top) and any resulting FRET efficiency (bottom).
(J) The dwell time of Cy3-MutS bound to a mismatch in the presence of ATP (200 μM; τMutS•mismatch•ATP = 4.2 ± 0.9 s, mean ± s.e.; n = 49).
(Adapted from W.-K. Cho et al. Structure. 20: 1264, 2012)


2. How does MMR develop access to an appropriate strand scission to initiate repair?

In E.coli the strand scission is provided by the MutH protein, which recognizes a hemimethylated GATC site and incises the unmethylated newly replicated strand. The directionality of the MutH strand scission insures that the repair of mismatched nucleotides arising from replication errors is confined to the DNA strand containing the mistake. Outside of gram-negative enteric bacteria the source of the strand scission and the mechanism for insuring faithful repair of the newly replicated strand are unknown. A potential source for the strand scission is the 3’ leading-strand or the 5’ lagging-strand ends of replicating DNA. However, these ends may be occupied by replication components. Similarly, chromatin assembly/disassembly obstacles will almost certainly be encountered during DDR signaling.


3. What are the differences in lesion sensing in chromatin compared to mismatch sensing for MMR?

The vast majority of biochemical studies have been performed with naked DNA, which is significantly different from the cellular chromatin where mismatch and lesion recognition occurs. Cellular chromatin is dynamic. This is especially true near the replication fork where MMR occurs, and where the fundamental units of chromatin, the nucleosomes, are being disassembled and then reassembled behind the replication machinery. The first fully formed nucleosome occurs ~250 bp behind the replication fork. Since the MMR excision tract may extend for several thousand nucleotides, the MMR machinery will almost certainly encounter both partial and fully formed nucleosomes. The histones that form the protein core of nucleosomes often contain post-translation modifications (PTMs) that regulate the dynamics of cellular chromatin. We have found that hMSH2-hMSH6 can disassemble nucleosomes adjacent to a mismatch, if the histones contain PTMs that increase the intrinsic nucleosome dynamics. In contrast, nucleosomes that are stably associated with nucleosome positioning sequences are refractory to disassembly and block repair. These results suggest a reciprocal interaction between MMR components and chromatin that may influence the choice between repair and DDR.


4. How do MMR proteins function to suppress recombination between non-allelic partially homologous DNA sequences during double-stranded break (DSB) repair?

The mechanism of HOER suppression by MMR is unknown and is especially important since tumor-associated chromosomal rearrangements often display the HOER signature.

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