Structural mechanism of DNA-PK allosteric activation in NHEJ pathway
Siyu is a 2nd year IBiS student in Yuan He's lab. He received a B.S. in Biological sciences from Peking University.
DNA double strand breaks (DSBs) is caused by either endogenous agents such as reactive oxygen species, or exogenous ionizing radiation and chemicals. DSBs are troublesome damages that are commonly found in the genome of cancerous cells, and incorrect DSB repair can potentially lead to undesired genome rearrangements, such as deletions, translocations, and fusions. DSBs can be repaired through either homologous recombination (HR), in which homologous DNA template is used to direst direct error-free repairs, or non-homologous end- joining (NHEJ) pathway, in which the two broken ends are directly ligated without referring to a homologous template. As a crucial DSB repair pathway, NHEJ is also used in V(D)J recombination, where various immunoglobulin genes are generated by exon recombination in immune cells. DNA break recognition in NHEJ requires DNA-dependent protein kinase (DNA- PK) holoenzyme, consisting of a flexible, ~470 kDa DNA-PK catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Years of efforts have been spent on investigating the individual components of this NHEJ initiation complex, but current structural information obtained by x- ray crystallography and cryo-electron microscope (cryo-EM) are limited by both resolution (>4Å) and complex integrity. Thus, the mechanism of how DNA-PK holoenzyme interacts with broken DNA end and subsequently recruit other DNA repair factors (Artemis, XRCC4, DNA ligase IV, XLF, and PAXX) in NHEJ pathway remains unclear.
Preliminary negative-staining EM experiments revealed a large conformational change of the N-terminal (cyan) arm domain of DNA-PKcs upon DNA binding. Therefore, I have come up with a hypothesis that the rearrangement of N-terminal domain in DNA-PKcs during complex formation could activate the kinase in a DNA-dependent manner, triggering the cascade of recruitment and modification of other NHEJ repair factors. However, the low-resolution model was not able to provide enough information about how this regulation event could occur. Since 2013, the newly developed electron direct detector has revolutionized our way to determine the structures of macromolecular machines using cryo-EM. The striking ability to directly visualize these large molecular complexes at near-atomic resolution makes cryo-EM an ideal technique for structure determination of this large DNA-PKcs/Ku70/Ku80/DNA complex (~620 kDa). Thus, the goal of my project is to use this method to determine the structure of this key NHEJ complex, and investigate the mechanisms used in DSB recognition and NHEJ repair pathway initiation.