Biology subjects

Hoitsma, N. M.

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

5 recordsLinked to original sources

bioRxiv2025

APE1 active site residue Asn174 stabilizes the AP-site and is essential for catalysis

Apurinic/Apyrimidinic (AP)-sites are common and highly mutagenic DNA lesions that can arise spontaneously or as intermediates during Base Excision Repair (BER). The enzyme apurinic/apyrimidinic endonuclease 1 (APE1) initiates repair of AP-sites by cleaving the DNA backbone at the AP-site via its endonuclease activity. Here, we investigated the functional role of the APE1 active site residue N174 that contacts the AP-site during catalysis. We analyzed the effects of three rationally designed APE1 mutations that alter the hydrogen bonding potential, size, and charge of N174: N174A, N174D, and N174Q. We found impaired catalysis of the APE1N174A and APE1N174D mutants due to disruption of hydrogen bonding and electrostatic interactions between residue 174 and the AP-site. In comparison, the APE1N174Q mutant was less impaired due to retaining similar hydrogen bonding and electrostatic characteristics as N174 in wild-type APE1. Structures and computational simulations further revealed that the AP-site was destabilized within the active sites of the APE1N174A and APE1N174D mutants due to loss of hydrogen bonding between residue 174 and the AP-site. Cumulatively, we show that N174 stabilizes the AP-site within the APE1 active site through hydrogen bonding and electrostatic interactions to enable effective catalysis. These findings highlight the importance of N174 in APE1s function and provide new insights into the molecular mechanism by which APE1 processes AP-sites during DNA repair.

biochemistry↗

bioRxiv2024

SMARCAD1 Regulates R-Loops at Active Replication Forks Linked to Cancer Mutation Hotspots

DNA replication often encounters obstacles like the stalled transcription machinery and R-loops. While ribonucleases and DNA-RNA helicases can resolve these structures, the role of chromatin remodelers remains understudied. Through a series of in vitro and in vivo experiments, we show that the chromatin remodeler SMARCAD1, which associates with active replication forks, is crucial for resolving nearby R-loops to maintain fork stability. SMARCAD1 directly binds R-loops via its ATPase domain and associates with the replisome through its N-terminus region. Both interactions are critical for resolving R-loops within cells. Genome-wide assays reveal that cells expressing mutant SMARCAD1 accumulate significantly more R-loops than wild-type cells, particularly in regions distinct from known fork blockage-prone sites. These R-loop-enriched regions in SMARCAD1 mutants also exhibit increased mutagenesis in germline tumors, suggesting they are mutation hotspots in cancer. Therefore, SMARCAD1 acts as an R-loop sensor and resolvase at actively progressing forks, maintaining genome stability and preventing tumorigenesis.

genetics↗

bioRxiv2024

Characterization of Medusavirus encoded histones reveals nucleosome-like structures and a unique linker histone

The organization of DNA into nucleosomes is a ubiquitous and ancestral feature that was once thought to be exclusive to the eukaryotic domain of life. Intriguingly, several representatives of the Nucleocytoplasmic Large DNA Viruses (NCLDV) encode histone-like proteins that in Melbournevirus were shown to form nucleosome-like particles. Medusavirus medusae (MM), a distantly related giant virus, encodes all four core histone proteins and, unique amongst most giant viruses, a putative acidic protein with two domains resembling linker histone H1. Here we report the structure of nucleosomes assembled with Medusavirus histones and highlight similarities and differences with eukaryotic and Melbournevirus nucleosomes. Our structure provides insight into how variations in histone tail and loop lengths are accommodated within the context of the nucleosome. We show that Medusavirus histones assemble into tri-nucleosome arrays, and that the putative linker histone H1 does not function in chromatin compaction. These findings expand our understanding of viral histones and suggest that Medusavirus histones represent a snapshot in the evolutionary timeline of nucleosome architecture. ONE SENTENCE SUMMARYThe four Medusavirus medusae core histones form nucleosome-like structures that combine features of eukaryotic and other viral nucleosomes.

molecular biology↗

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↗