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The Structure of the Nucleosomal DNA Repair Intermediate Affects the HPF1-Independent Automodification Activity of
Tatyana A Kurgina1, Danil M Shtanov1,2, Mikhail M Kutuzov1
1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.
Abstract:
DNA-dependent nuclear enzymes poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) are involved in the regulation of multiple DNA repair pathways, including base excision repair (BER). After activation by binding to damaged DNA, these enzymes synthesize negatively charged poly(ADP-ribose) (PAR) and covalently attach to amino acid residues of target proteins, including PARPs themselves. PARP2 activity is influenced by the nature of DNA lesion; for example, it is efficiently stimulated by DNA breaks flanked by phosphate group. However, it remains unclear which stages of the auto-PARylation reaction are most sensitive to the structure of damaged DNA. In this study, we investigated how PARP2 activity depends on the presence and position of a single-nucleotide gap in DNA (either free or in the context of nucleosome) at different stages of the automodification reaction conducted in the absence of the histone PARylation factor HPF1. The obtained results suggest that the presence of the gap affects the affinity of PARP2 for DNA/nucleosomes, thereby determining the number of catalytically active enzyme molecules and the efficiency of PARylation initiation. In contrast, PAR elongation was affected by the lesion location in the DNA/nucleosome structure, namely, its distance from the blunt DNA ends, and the environment of histone tails. Therefore, the damaged DNA structure can influence both the amount and the length of PAR synthesized by PARP2.
Insights
Poly(ADP-ribose) polymerase 2 (PARP2) activity is sensitive to DNA damage structure. DNA gaps affect PARP2 binding and initiation, while lesion location influences PAR chain elongation during DNA repair.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Poly(ADP-ribose) polymerases (PARP1 and PARP2) are key nuclear enzymes regulating DNA repair pathways like base excision repair (BER).
- PARP enzymes are activated by damaged DNA, synthesizing poly(ADP-ribose) (PAR) chains that modify target proteins.
- PARP2 activity is known to be influenced by DNA lesion characteristics, but the precise impact of DNA structure on its auto-PARylation stages remains unclear.
Purpose of the Study:
- To investigate the impact of single-nucleotide DNA gaps on PARP2 activity at various auto-PARylation stages.
- To determine how DNA gap presence and location, within free DNA or nucleosomes, affect PARP2 function.
- To elucidate the role of DNA structure and histone environment in modulating PARP2-mediated PARylation.
Main Methods:
- Enzymatic assays measuring PARP2 activity on DNA substrates with single-nucleotide gaps.
- Experiments conducted on both free DNA and DNA within nucleosome structures.
- Analysis of PARP2 auto-PARylation initiation and elongation in the absence of Histone PARylation Factor 1 (HPF1).
Main Results:
- The presence of a DNA gap influenced PARP2's affinity for DNA/nucleosomes, affecting the number of active enzyme molecules and PARylation initiation efficiency.
- PAR chain elongation by PARP2 was sensitive to the lesion's location within the DNA/nucleosome structure, specifically its proximity to blunt ends and histone tail interactions.
- These findings indicate that DNA structural features significantly modulate both the quantity and length of PAR synthesized by PARP2.
Conclusions:
- DNA damage structure plays a critical role in regulating PARP2 activity during DNA repair.
- PARP2's response to DNA lesions involves distinct sensitivities at different stages of the auto-PARylation reaction.
- The findings provide insights into how DNA context influences PARP enzyme function in maintaining genomic integrity.
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