Biology subjects

Hoitsma, N. M.

Publications and source records attributed to Hoitsma, N. M..

2 recordsLinked to original sources

bioRxiv2022

Mechanistic Insight into AP-Endonuclease 1 Cleavage of Abasic Sites at Stalled Replication Forks

1.Many types of DNA damage stall replication fork progression, including abasic sites. AP-Endonuclease 1 (APE1) has been shown to cleave abasic sites in ssDNA substrates. Importantly, APE1 cleavage of ssDNA at a replication fork has significant biological implications by generating double strand breaks that could collapse the replication fork. Despite this, the molecular basis and efficiency of APE1 processing abasic sites at a replication fork remains elusive. Here, we investigate APE1 cleavage of several abasic substrates that mimic potential APE1 interactions at replication forks. We determine that APE1 has robust activity on these substrates, similar to dsDNA, and report rapid rates for cleavage and product release. X-ray crystal structures visualize the APE1 active site, highlighting that a similar mechanism is used to process ssDNA substrates as canonical APE1 activity on dsDNA. However, mutational analysis reveals R177 to be uniquely critical for the APE1 ssDNA cleavage mechanism. Additionally, we investigate the interplay between APE1 and Replication Protein A (RPA), the major ssDNA-binding protein at replication forks, revealing that APE1 can cleave an abasic site while RPA is still bound to the DNA substrate. Together, this work provides molecular level insights into abasic ssDNA processing by APE1, including the presence of RPA.

biochemistry↗

bioRxiv2022

Structural basis for APE1 processing DNA damage in the nucleosome

Genomic DNA is continually exposed to endogenous and exogenous factors that promote DNA damage. Eukaryotic genomic DNA is packaged into nucleosomes, which present a barrier to accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome and protect genomic stability is unknown. Here, we determine how the base excision repair (BER) endonuclease AP-endonuclease 1 (APE1) recognizes and cleaves DNA damage in the nucleosome. Kinetic assays determined that APE1 cleaves solvent-exposed AP sites in the nucleosome with 3 - 6 orders of magnitude higher efficiency than occluded AP sites. A cryo-electron microscopy structure of APE1 bound to a nucleosome containing a solvent-exposed AP site identified that APE1 uses a DNA sculpting mechanism for AP site recognition, where APE1 bends the nucleosomal DNA to access the AP site. Notably, additional biochemical and structural characterization of occluded AP sites identified contacts between the nucleosomal DNA and histone octamer that prevent efficient processing of the AP site by APE1. These findings provide a rationale for the position-dependent activity of BER proteins in the nucleosome and suggests the ability of BER proteins to sculpt nucleosomal DNA drives efficient BER in chromatin.

biochemistry↗