Genome stability and integrity are essential for survival of all organisms. However, due to the endogenous metabolites and environmental agents, DNA damage constantly happens and posts a threat.

DNA glycosylase is one DNA-binding protein responsible for repairing the DNA damage. It remains a puzzle how glycosylase can efficiently and accurately recognize DNA lesions among the millions or billions of normal base pairs in the genome.

It has been hypothesized that glycosylase accomplish this task by alternating between two diffusion modes: one high-speed-low-accuracy mode and one low-speed-high accuracy mode. However, due to the limitation in the spatial and temporal resolutions of current experimental techniques, the slow mode has not been successfully detected. 

In order to understand the molecular mechanism how glycosylase AlkD recognizes the DNA damage, Prof. ZHANG Lu from Fujian Institute of Research on the Structure of Matter of Chinese Academy of Sciences and his collaborators have integrated experimental and computational methods to characterize the dynamic diffusion of glycosylase AlkD along double-stranded DNA (dsDNA) at molecular level.

The study was published in Proceedings of the National Academy of Sciences of the United States of America on August 20.

The researchers developed a scanning Fluorescence Resonance Energy Transfer – Fluorescence Correlation Spectroscopy platform to provide protein dynamics at the microsecond temporal resolution and sub-nanometer spatial resolution.  

The significant improvement in the resolution enables the researchers not only observe the fast mode, but also directly capture the slow mode.   

To further elucidate the underlying molecular mechanism of the slow mode, the researchers constructed Markov State Model (MSM) based on extensive all-atom molecular dynamics (MD) simulations.  

Based on MSM, the researchers visualized continuous cycles of AlkD diffusion along dsDNA over 1ms, the timescale of which is difficult to be reached by conventional MD simulations. They revealed that the diffusion of AlkD over one base pair contained a rate-limiting rotation and a sequential translation.  

Moreover, they pinpointed the essential role of Y27 in determining the AlkD diffusion dynamics both experimentally and computationally.  

The study provided mechanistic insights on how conformational dynamics of AlkD-dsDNA complex coordinate different diffusion modes to recognize DNA lesions with high efficiency and accuracy.