Damage search mechanism of human NER protein

Abstract

NER (nucleotide excision repair) is one of DNA repair mechanisms that amend DNA damage such as UV-induced thymine dimers or chemical modifications on bases. The failure of NER causes genetic diseases such as xeroderma pigmentosum and Cockayne syndrome. [1] Human XPC-Rad23B (hXPC-Rad23B) complex is the first protein to initiate the entire global genome NER process by recognizing DNA lesions. However, the damage search mechanism of hXPC-Rad23B remains poorly understood. Here, we investigated the damage search process of hXPC-Rad23B using the single-molecule DNA curtain technique, which is a novel imaging technique concatenating microfluidics, lipid fluidity, and fluorescence microscopy. [2] We observed the movement of hXPC-Rad23B on undamaged lambda DNA and found that hXPC-Rad23B diffuses along DNA, indicating that hXPC-Rad23B finds DNA damage through one-dimensional diffusion. In addition, some hXPC-Rad23B molecules stably stay on DNA and others exhibits sub-diffusive motion within ~3 kbp. The stationary behavior is assigned as immobile motion and the sub-diffusive fluctuation is designated as constrained motion. Surprisingly, the position where the constrained and immobile motions occur is highly correlated with the position of the consecutive AT-tracks in the lambda DNA. In the AT-track regions, the rapid opening and closing of duplex DNA, called as DNA bubbling (or breathing), frequently occur. Our results suggest that the constrained motion is due to the DNA bubbling in the AT-track regions. Moreover, we analyzed the diffusion coefficient of hXPC-Rad23B, which dramatically rises up with increasing salt concentration. This demonstrates that hXPC-Rad23B finds DNA damage through the hopping mechanism.