Abstract
DNA double strand breaks (DSBs) can be generated by endogenous cellular processes or exogenous agents in mammalian cells. These breaks are highly variable with respect to DNA sequence and structure and all are recognized in some context by the DNA-dependent protein kinase (DNA-PK). DNA-PK is a critical component necessary for the recognition and repair of DSBs via non-homologous end joining (NHEJ). Previously studies have shown that DNA-PK responds differentially to variations in DSB structure, but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanisms by which DNA-PK is activated by various DNA substrates and provide evidence that the DNA-PK is differentially activated by DNA structural variations as a function of the C-terminal region of Ku80. Discrimination based on terminal DNA sequence variations, on the other hand, is independent of the Ku80 C-terminal interactions and likely results exclusively from DNA-dependent protein kinase catalytic subunit interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. These results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately the efficiency of DSB repair.
Highlights
Efficient repair of DNA double strand breaks (DSBs) requires the recognition and resolution of a wide variety of DNA termini including variations in structure, DNA sequence, and chemistry
To determine if the Ku80 C-terminus plays a role in DNA discrimination by DNA-PK, a series of Ku80 C-terminal truncation constructs were constructed based on the Ku 80 structural features and domains (Fig 1A)
Our results establish distinct mechanisms by which DNA-PK is able to respond to a variety of DNA substrates to support repair
Summary
Efficient repair of DNA DSBs requires the recognition and resolution of a wide variety of DNA termini including variations in structure, DNA sequence, and chemistry. The DNA-dependent protein kinase (DNA-PK) is a heterotrimer consisting of Ku70, Ku80, and the catalytic subunit (DNA-PKcs). DNA-PK initiates the repair of the preponderance of DSB in mammalian cells and regulates the Non-Homologous End Joining Pathway (NHEJ) through its kinase activity. DNA-PK activation requires both protein/protein interactions within the heterotrimer and protein/DNA interactions, which are not mutually exclusive [1,2]. DNA-PK phosphorylates several important targets including p53, RPA, and histone H2AX [3,4,5,6,7,8]. We have previously reported that the DNA to which DNA-PK is bound modulates
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