Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro.

The ability of cell-free extracts to correct DNA mismatches has been demonstrated in both prokaryotes and eukaryotes. Such an assay requires a template containing both a mismatch and a strand discrimination signal, and the multi-step construction process can be technically difficult. We have developed a three-step procedure for preparing DNA heteroduplexes containing a site-specific nick. The mismatch composition, sequence context, distance to the strand signal, and the means for assessing repair in each strand are adjustable features built into a synthetic oligonucleotide. Controlled ligation events involving three of the four DNA strands incorporate the oligonucleotide into a circular template and generate the repair-directing nick. Mismatch correction in either strand of a prototype G.T mismatch was achieved by placing a nick 10-40 bp away from the targeted base. This proximity of nick and mismatch represents a setting where repair has not been well characterized, but the presence of a nick was shown to be essential, as was the MSH2/MSH6 heterodimer, although low levels of repair occurred in extract defective in each protein. All repair events were inhibited by a peptide that interacts with proliferating cell nuclear antigen and inhibits both mismatch repair and long-patch replication.

Strand-specific processing of 8-oxoguanine by the human mismatch repair pathway: inefficient removal of 8-oxoguanine paired with adenine or cytosine.

Genomic DNA and its precursors are susceptible to oxidation during aerobic cellular metabolism, and at least five distinct repair activities target a single common lesion, 7,8-dihydro-8-oxoguanine (8-oxoG). The human mismatch repair (MMR) pathway, which has been implicated in an apoptotic response to covalent DNA damage, is likely to encounter 8-oxoG in both the parental and daughter strand during replication. Here, we show that lesions containing 8-oxoG paired with adenine or cytosine, which are most likely to arise during replication, are not efficiently processed by the mismatch repair system. Lesions containing 8-oxoG paired with thymine or guanine, which are unlikely to arise, are excised in an MSH2/MSH6-dependent manner as effectively as the corresponding mismatches when placed in a context that reflects the daughter strand during replication. Using a newly developed assay based on methylation sensitivity, we characterized strand-excision events opposite 8-oxoG situated to reflect placement in the parental strand. Lesions that efficiently trigger strand excision and resynthesis (8-oxoG paired with thymine or guanine) result in adenine or cytosine insertion opposite 8-oxoG. These latter pairings are poor substrates for further action by mismatch repair, but precursors for alternative pathways with non-mutagenic outcomes. We suggest that the lesions most likely to be encountered by the human mismatch repair pathway during replication, 8-oxoG.A or 8-oxoG.C, are likely to escape processing in either strand by this system. Taken together, these data suggest that the human mismatch repair pathway is not a major contributor to removal of misincorporated 8-oxoG, nor is it likely to trigger repeated attempts at lesion processing.

Constraining protein sequence space: four amino acid alphabets are sufficient to recapitulate lambda repressor multimerization.

Nucleic acid polymers selected from random sequence space constitute an enormous array of catalytic, diagnostic and therapeutic molecules. Despite the fact that proteins are robust polymers with far greater chemical and physical diversity, success in unlocking protein sequence space remains elusive. We have devised a combinatorial strategy for accessing nucleic acid sequence space corresponding to proteins comprising selected amino acid alphabets. Using the SynthOMIC approach (synthesis of ORFs by multimerizing in-frame codons), representative libraries comprising four amino acid alphabets were fused in-frame to the lambda repressor DNA-binding domain to provide an in vivo selection for self-interacting proteins that re-constitute lambda repressor function. The frequency of self-interactors as a function of amino acid composition ranged over five orders of magnitude, from approximately 6% of clones in a library comprising the amino acid residues LARE to approximately 0.6 in 10(6) in the MASH library. Sequence motifs were evident by inspection in many cases, and individual clones from each library presented substantial sequence identity with translated proteins by BLAST analysis. We posit that the SynthOMIC approach represents a powerful strategy for creating combinatorial libraries of open reading frames that distils protein sequence space on the basis of three inherent properties: it supports the use of selected amino acid alphabets, eliminates redundant sequences and locally constrains amino acids.

DNA template requirements for human mismatch repair in vitro.

The human mismatch repair pathway is competent to correct DNA mismatches in a strand-specific manner. At present, only nicks are known to support strand discrimination, although the DNA end within the active site of replication is often proposed to serve this role. We therefore tested the competence of DNA ends or gaps to direct mismatch correction. Eight G.T templates were constructed which contained a nick or gap of 4, 28, or approximately 200 nucleotides situated approximately 330 bp away in either orientation. A competition was established in which the mismatch repair machinery had to compete with gap-filling replication and ligation activities for access to the strand discontinuity. Gaps of 4 or 28 nucleotides were the most effective strand discrimination signals for mismatch repair, whereas double strand breaks did not direct repair to either strand. To define the minimal spatial requirements for access to either the strand signal or mismatch site, the nicked templates were linearized close to either site and assayed. As few as 14 bp beyond the nick supported mismatch excision, although repair synthesis failed using 5'-nicked templates. Finally, asymmetric G.T templates with a remote nick and a nearby DNA end were repaired efficiently.

DNA bending and unbending by MutS govern mismatch recognition and specificity.

DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base-base mismatches and insertion/deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS-mismatch complex but not in the MutS-homoduplex complex that sets off the cascade of events that leads to repair. To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS-DNA complexes by using atomic force microscopy. Interestingly, MutS-DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites. These results suggest that the specific recognition complex is one in which the DNA is unbent. Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (i) binds to DNA nonspecifically and bends it in search of a mismatch; (ii) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (iii) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent. Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity.