Facilitated Diffusion Mechanisms in DNA Base Excision Repair and Transcriptional Activation

Abstract

Preservation of the coding potential of the genome and highly regulated gene expression over the life span of a human are two fundamental requirements of life. These processes require the action of repair enzymes or transcription factors that efficiently recognize specific sites of DNA damage or transcriptional regulation within a restricted time frame of the cell cycle or metabolism. A failure of these systems to act results in accumulated mutations, metabolic dysfunction, and disease. Despite the multifactorial complexity of cellular DNA repair and transcriptional regulation, both processes share a fundamental physical requirement that the proteins must rapidly diffuse to their specific DNA-binding sites that are embedded within the context of a vastly greater number of nonspecific DNA-binding sites. Superimposed on the needle-in-the-haystack problem is the complex nature of the cellular environment, which contains such high concentrations of macromolecules that the time frame for diffusion is expected to be severely extended as compared to dilute solution. Here we critically review the mechanisms for how these proteins solve the needle-in-the-haystack problem and how the effects of cellular macromolecular crowding can enhance facilitated diffusion processes. We restrict the review to human proteins that use stochastic, thermally driven site-recognition mechanisms, and we specifically exclude systems involving energy cofactors or circular DNA clamps. Our scope includes ensemble and single-molecule studies of the past decade or so, with an emphasis on connecting experimental observations to biological function.

Direct Observation and Analysis of the Dynamics of the Photoresponsive Transcription Factor GAL4

“…GAL4–VVD moved on the five consecutive UASG sites, without specifically binding to these sites, for 230 s (frame 46 in Figure b), and then decelerated and fluctuated around site I (Figure b). Furthermore, long distance transfer, such as fast sliding or hopping along the dsDNA, occurred . We also observed multiple traces of GAL4–VVD in the same AFM image.…”

“…During stalling, two DNA‐binding domains in recognition mode were used for complex formation at the specific site and for subsequent relocation within these sites (as observed after frame 36 in Figure d). On the other hand, during the sliding movement, DNA‐binding domains with a conformation suitable for searching would have been used for the sliding (as observed in frames 1–35 in Movie S4 in the Supporting Information), similar to that observed for the other Zn‐finger protein . By visualizing the movement of GAL4–VVD at nanoscale resolution in the DNA frame, these two distinct modes, termed searching mode and recognition mode, could be identified.…”

“…Transcription factors function as molecular switches to regulate transcription; this dynamic behavior involving DNA needs further elucidation to enable the understanding of the switching mechanism. Transcription factors and enzymes have been directly observed and several searching mechanisms for target sites, including sliding, hopping, intersegmental transfer, and three‐dimensional diffusion, have been proposed . The dynamics of DNA‐binding proteins during the search for target sites has been mainly studied using fluorescence microscopy at sub‐micrometer resolution .…”

“…The overall 1D diffusion coefficient in the sliding was calculated to be D =6.7×10 1 nm 2 s −1 . Due to the interaction with the mica surface, the 1D diffusion coefficient obtained in this experiment was two orders of magnitude smaller than the reported values for DNA‐binding proteins in solution . We also observed fast sliding or hopping, during which GAL4–VVD moved more than 100 bp/frame.…”

“…We also observed fast sliding or hopping, during which GAL4–VVD moved more than 100 bp/frame. For the long‐distance transfer, hopping is advantageous, because proteins can rapidly move along the dsDNA for long distance via dissociative transfer to search for a specific site . Two Zn‐finger DNA‐binding domains work asymmetrically to realize rapid transfer during sliding .…”