Base excision repair in nucleosomes lacking histone tails

Area of Science:

• Molecular Biology
• Epigenetics
• DNA Repair

Background:

• Chromatin structure, specifically nucleosomes, can impede DNA repair processes.
• Base excision repair (BER) is crucial for maintaining genomic integrity.
• Histone N-terminal tails play roles in nucleosome interactions and are sites of post-translational modifications.

Purpose of the Study:

• To investigate the impact of histone N-terminal tails on BER enzyme activity within a designed nucleosome.
• To determine if removing histone tails alleviates inhibition of BER enzymes, particularly DNA polymerase beta (pol beta).
• To assess the effect of XRCC1 and WRN proteins on BER in the presence of histone tails.

Main Methods:

• Development of an in vitro system using human uracil DNA glycosylase (UDG), AP endonuclease (APE), and pol beta.
• Construction of rotationally positioned nucleosomes with a single uracil or G:U mismatch.
• Limited trypsin digestion to remove histone tails from nucleosomes and histone octamers.
• Exchange of histone octamers onto modified DNA to create tailless nucleosomes.
• Assay of BER enzyme activities on both intact and tailless nucleosomes, with and without XRCC1/WRN.

Main Results:

• UDG and APE showed reduced efficiency on nucleosomes, with activity dependent on uracil's rotational orientation.
• Removal of histone tails did not significantly enhance UDG or APE activity.
• Pol beta activity remained completely inhibited on nucleosomes even after histone tail removal.
• XRCC1 and WRN proteins did not restore BER activity on nucleosomal DNA.

Conclusions:

• Histone N-terminal tails are not the primary cause of pol beta inhibition within nucleosomes.
• Specific DNA repair factors may be required for efficient BER in chromatin.
• Nucleosome structure itself, beyond histone tails, significantly impacts BER enzyme function.