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Updated: Jan 19, 2026
Author Spotlight: Efficient Nucleosome Reconstitution for Single-Molecule Techniques
Published on: September 6, 2024
DNA-Protein Cross-Link Formation in Nucleosome Core Particles Treated with Methyl Methanesulfonate
1Department of Chemistry , Johns Hopkins University , 3400 N. Charles Street , Baltimore , Maryland 21218 , United States.
Abstract:
N7-Methyl-2'-deoxyguanosine (MdG) is the major damage product in DNA produced by methylating agents, but it often thought to be nontoxic and nonmutagenic. MdG is chemically unstable. An abasic site (AP) is the major product produced from MdG under physiologically relevant conditions. AP formation is frequently considered to be responsible for the cytotoxic effects of MdG, but the reaction is suppressed in nucleosome core particles (NCPs). Recently, it was discovered that histone proteins form reversible DNA-protein cross-links (DPCs) with MdG in reconstituted NCPs, as well as in methylmethanesulfonate (MMS) treated cells. In this study, the formation and reactivity of MdG in MMS treated NCPs was examined at single nucleotide resolution. Sequences consisting of three or more consecutive dGs are more reactive with MMS. The efficiency and selectivity of MdG formation by MMS is largely unaffected within a NCP, although reactivity at several dGs is ∼1.5-2.5-fold higher in NCPs. DPC formation from MdG (DPCMdG) predominates over AP at all positions within the NCP. With few exceptions, DPCMdG yield is strongly dependent upon the accessibility of the major groove containing MdG to lysine-rich histone N-terminal tails. These data indicate that histone-MdG DPC formation will depend upon DNA sequence and translational position within an NCP.
Insights
Histone proteins form DNA-protein cross-links (DPCs) with N7-methyl-2'-deoxyguanosine (MdG) in nucleosome core particles (NCPs), a reaction that predominates over abasic site formation and depends on DNA sequence and histone accessibility.
Area of Science:
- Molecular Biology
- DNA Damage and Repair
- Epigenetics
Background:
- N7-methyl-2'-deoxyguanosine (MdG) is a primary DNA lesion from methylating agents.
- MdG is chemically unstable, often yielding abasic (AP) sites, linked to cytotoxicity.
- AP formation is suppressed within nucleosome core particles (NCPs).
Purpose of the Study:
- Investigate MdG formation and reactivity within NCPs at single nucleotide resolution.
- Determine the factors influencing MdG processing in a chromatin context.
- Clarify the role of DNA-protein cross-links (DPCs) in MdG lesion fate.
Main Methods:
- Reconstitution of NCPs with specific DNA sequences.
- Treatment with methylmethanesulfonate (MMS) to induce DNA methylation.
- Single nucleotide resolution analysis of MdG formation and DPC/AP site generation.
- Assessment of histone N-terminal tail interactions with MdG.
Main Results:
- Sequences with ≥3 consecutive guanines show higher MMS reactivity.
- MdG formation efficiency in NCPs is similar to naked DNA, but reactivity is 1.5-2.5x higher at specific sites.
- DNA-protein cross-link formation (DPCMdG) is favored over AP site formation within NCPs.
- DPCMdG formation is dependent on the accessibility of the major groove to histone tails.
Conclusions:
- Histone-MdG DPC formation is a significant pathway in NCPs, competing with AP site formation.
- The formation of these DPCs is influenced by DNA sequence and the positioning of MdG within the NCP.
- Histone interactions play a critical role in modulating the fate of MdG lesions within chromatin.
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