단일분자 기술을 이용한 염기쌍 오류복구 단백질 연구

Abstract

The mismatch repair system is a well-conserved process which maintains the genetic stability in extremely high fidelity during DNA replication both in the eukaryote and prokaryote. In the mismatch repair system MutS protein initially recognizes a mismatch on DNA and transmits the mismatch finding to the distant strand scission site for a re-synthesis of error-contained strand via interactions with downstream repair proteins. The involved proteins for the mismatch repair are well-characterized for several decades, but the definite mechanisms of sub-steps in this process is still controversial. Using single-molecule föster resonance energy transfer (smFRET), we determined that MutS forms a transient clamp that scans duplex DNA for mismatched nucleotide by 1D diffusion, and then the mismatch recognition by MutS provokes ATP binding and alters the conformation of MutS to a distinctly different diffusive clamp with high stability on DNA, which may be released by single-stranded DNA. To confirm these observed processes and to image MutS diffusion dynamics on relatively long DNA containing a single mismatch, we combined the real-time fluorescent particle tracking method with FRET. Along with another assay using polarized total internal reflection fluorescence microscope, which allows us to investigate the rotational property of MutS, we revealed that searching MutS rotates during diffusion independent of ionic strength or an external drag force, which suggests continuous contact with the DNA backbone. In contrast, ATP-bound MutS clamps spin freely around the DNA, and their diffusion is affected by ionic strength and a drag force. To address the interplay between DNA replication and DNA mismatch repair, we also studied the physical interaction between MutS and the DNA replication holoenzyme beta-subunit, a sliding clamp that enhances the processivity of DNA polymerase. The beta-clamp recruits MutS on duplex DNA through a direct interaction. After MutS with beta-clamp is placed at the mismatch, beta-clamp is released from the MutS bound to the mismatch. The ATP-bound MutS does not seem to interact with beta-clamp diffusing on DNA.