Structural basis of end processing in the nucleosome by polynucleotide kinase phosphatase

Genomic DNA is intricately packaged into chromatin through the nucleosome core particle, which is a fundamental repeating unit. This chromatinized DNA is constantly exposed to various forms of damage from both internal and external sources, making effective repair crucial to maintain genomic stability. Single-strand breaks (SSBs) with chemically diverse DNA ends represent one of the most common types of genomic DNA damage.

To repair these SSBs, they must first be processed enzymatically before undergoing gap-filling DNA synthesis or nick ligation during single-strand break repair (SSBR). Polynucleotide kinase phosphatase (PNKP) is a versatile enzyme involved in this end-processing task, possessing two essential catalytic activities. However, the precise mechanism of how PNKP operates within the chromatin environment to initiate SSBR has remained unclear until now.

This study combines biochemical assays and cryogenic electron microscopy (cryo-EM) to elucidate the molecular basis of end processing in the nucleosome by PNKP. Cryo-EM structures of PNKP interacting with non-ligatable SSBs at three distinct positions within the nucleosome reveal that PNKP deforms the nucleosomal DNA to reposition these SSBs into the kinase and phosphatase active sites.

This structural insight offers a compelling explanation for the efficient processing of SSBs throughout the nucleosome. Additionally, cryo-EM structures demonstrate that the PNKP FHA domain also interacts with the nucleosome acidic patch during non-ligatable SSB recognition, further accelerating the processing of these SSBs within the nucleosome.

These findings collectively provide valuable mechanistic insights into the initial end processing step of chromatin-based SSBR, paving the way for a deeper understanding of genomic stability maintenance.