How DNA-repair proteins find their targets

Abstract

Genomic DNA is subject to damage at such high frequencies that only with efficient DNArepair pathways genomic stability is maintained. Research efforts of many groups over several decades have revealed the salient properties of the various DNA-repair mechanisms, but it has largely remained unclear how damage sites are localized in genomic DNA. In PNAS, Gorman et al. (1) unveil insights into the DNA-repair process called mismatch repair (MMR), which is responsible for the repair of incorrectly paired DNA bases. They use a combination of singlemolecule fluorescence imaging and nanofabrication to study how different search mechanisms are used in eukaryotic MMR to find mismatches.The very first step of any attempt to repair damage that is highly localized to one specific position on the DNA, most often to just one base, consists of the formidable task of finding this site amid billions of bases of undamaged DNA. Often time constraints also play an important role, for example when damage needs to be corrected before replication takes place to prevent stalling of the replication machinery, or the errors irreversibly being propagated to progeny cells. Solely relying on 3D diffusion has long been thought to be insufficient for the DNA-repair process to win this race against the clock. It was proposed a number of decades ago that the search of a protein for a specific site on DNA (be it a repair protein finding lesions, transcription factor binding to promoters, or any other DNA-binding protein that needs to associate with a unique and specific position) could be drastically sped up by combining 3D diffusion through solution with short stretches of one-dimensional (1D) search along DNA (2). In such a picture, the protein would randomly bind to a position on DNA from solution, scan around the DNA in a 1D, random walk-like fashion, dissociate to rebind at another position, and repeat this cycle until the target site is found.Several molecular scenarios have been proposed, and experimentally confirmed, that describe the molecular details of how a protein can move along DNA in a 1D fashion. The first, sliding, can be best described as the protein scanning the DNA while maintaining continuous electrostatic contact with the backbone charges of the duplex. In such a scenario, the protein is rapidly moving back and forth while tracking the helical pitch of the DNA and thus rotating around the DNA. The second, hopping, involves the protein undergoing many rapid cycles of microscopic association and dissociation events onto and from the DNA. Although the protein does not move along the DNA during the fleeting moments it is bound to the DNA, it will be subject to 3D diffusion while transiently unbound and thus will likely rebind at a slightly different position. Many of such cycles in rapid succession will also cause the DNA to search onedimensionally along the DNA in a random walk-like fashion. Finally, it has been proposed that many such DNA-searching proteins may switch DNA strands during their search. By binding to another, nea...

Prerecognition Diffusion Mechanism of Human DNA Mismatch Repair Proteins along DNA: Msh2-Msh3 versus Msh2-Msh6

“…Msh2-Msh6 Chimera. Brown et al 24 performed an experiment to elucidate the role of MBDs in roadblock bypassing. They prepared a chimeric version of Msh2-Msh6 in which MBD6 was replaced by MBD3, which demonstrated that MBD3 imparts roadblock bypass activity to chimeric Msh2-Msh6.…”

“…Real-time single-molecule fluorescence resonance energy transfer (smFRET) studies showed that, in its search mode, MutS forms a clamp on the DNA. Changes in FRET efficiency and distribution with length and time resolution indicated that MutS carries out mismatch search by translation-coupled rotation diffusion while in continuous contact with the duplex DNA. , Using total internal reflection fluorescence microscopy (TIRFM), MutSα was also observed to perform translation-coupled rotation diffusion to maintain a constant register with the helical contour of the DNA. − …”