Comparison of the Base Excision and Direct Reversal Repair Pathways for Correcting 1,N⁶-Ethenoadenine in Strongly Positioned Nucleosome Core Particles

Abstract

1,N 6 -ethenoadenine (εA) is a mutagenic lesion and biomarker observed in numerous cancerous tissues. Two pathways are responsible for its repair: base excision repair (BER) and direct reversal repair (DRR). Alkyladenine DNA glycosylase (AAG) is the primary enzyme that excises εA in BER, generating stable intermediates that are processed by downstream enzymes. For DRR, the Fe(II)/α-ketoglutarate-dependent ALKBH2 enzyme repairs εA by direct conversion of εA to A. While the molecular mechanism of each enzyme is well understood on unpackaged duplex DNA, less is known about their actions on packaged DNA. The nucleosome core particle (NCP) forms the minimal packaging unit of DNA in eukaryotic organisms and is composed of 145−147 base pairs wrapped around a core of eight histone proteins. In this work, we investigated the activity of AAG and ALKBH2 on εA lesions globally distributed at positions throughout a strongly positioned NCP. Overall, we examined the repair of εA at 23 unique locations in packaged DNA. We observed a strong correlation between rotational positioning of εA and AAG activity but not ALKBH2 activity. ALKBH2 was more effective than AAG at repairing occluded εA lesions, but only AAG was capable of full repair of any εA in the NCP. However, notable exceptions to these trends were observed, highlighting the complexity of the NCP as a substrate for DNA repair. Modeling of binding of the repair enzymes to NCPs revealed that some of these observations can be explained by steric interference caused by DNA packaging. Specifically, interactions between ALKBH2 and the histone proteins obstruct binding to DNA, which leads to diminished activity. Taken together, these results support in vivo observations of alkylation damage profiles and contribute to our understanding of mutational hotspots.

Nucleosome Core Particles Lacking H2B or H3 Tails Are Altered Structurally and Have Differential Base Excision Repair Fingerprints

“…In this work, we compare the global repair profiles of AAG on εA lesions distributed throughout native NCPs or those lacking H2B or H3 tails. As observed previously, εA removal by AAG is highly correlated with the rotational orientation in the central region of the DNA in native NCPs. , We also reported a similar trend for the glycosylases 8-oxo-7,8-dihydroguanine glycosylases (OGG1), , uracil DNA glycosylase (UDG), , and thymine DNA glycosylase (TDG) . A correlation between the rotational orientation of a lesion and glycosylase activity has also been reported by other groups. − …”

“…εA lesions were incorporated throughout the “I” strand of the Widom 601 sequence to create a population of DNA containing εA:T base pairs, utilizing methods we reported previously. − , (The “I” strand designation is based on nomenclature used in the reported crystal structure of the 601 NCP …”

“…MID sites demonstrate much more variability, with most NCP/DUP values ranging between 0.1 and 0.6, except that of site 105, which reaches 1. This correlation between rotational orientation and excision is in agreement with previous reports. , …”

“…36 εA lesions were incorporated throughout the "I" strand of the Widom 601 sequence to create a population of DNA containing εA:T base pairs, utilizing methods we reported previously. [28][29][30]37 (The "I" strand designation is based on nomenclature used in the reported crystal structure of the 601 NCP. 36 ) Briefly, a Poisson distribution was used to determine the molar ratio of A to εA phosphoramidite building blocks used during chemical synthesis such that 95% of the synthesized DNA contain no more than a single εA.…”

“…This correlation between rotational orientation and excision is in agreement with previous reports. 28,37 Excision of εA from gH2B. For the majority of εA lesions, excision is comparable or enhanced in gH2B compared to native NCPs (Figure 4, red bars, and Table 1).…”