Arsenite retards DNA break rejoining by inhibiting DNA ligation

Area of Science:

• Molecular Biology
• Toxicology
• Biochemistry

Background:

• Arsenic (As) is known to interfere with methyl methane-sulphonate (MMS)-induced DNA repair, but the precise mechanism is debated.
• Understanding how As affects DNA repair is crucial for assessing its genotoxicity and developing mitigation strategies.

Purpose of the Study:

• To pinpoint the specific DNA repair step most vulnerable to arsenite (As) inhibition.
• To elucidate the molecular mechanism by which As disrupts DNA repair processes.

Main Methods:

• Single-cell alkaline electrophoresis was used to assess DNA strand breaks and rejoining.
• Hydroxyurea (Hu) plus cytosine-beta-D-arabinofuranoside (AraC) were employed to block DNA polymerization and accumulate strand breaks.
• In vitro assays with cell extracts compared the sensitivity of various DNA repair steps (excision, polymerization, ligation, synthesis) to As.
• The effect of dithiothreitol on As inhibition was investigated to explore the role of dithiol groups.

Main Results:

• As treatment increased DNA strand breaks in MMS-treated cells, suggesting excision is less sensitive than later steps.
• As showed weak inhibition of DNA strand break accumulation but significantly inhibited the rejoining of breaks.
• In vitro assays revealed the order of As inhibitory potency: ligation of poly(rA).oligo(dT) > ligation of poly(dA).oligo(dT) ≈ DNA polymerization ≥ DNA repair synthesis > excision.
• Dithiothreitol reversed As inhibition of poly(rA).oligo(dT) ligation and pyruvate dehydrogenase activity, but not poly(dA).oligo(dT) ligation.

Conclusions:

• Arsenite (As) primarily inhibits the DNA ligation step, specifically the rejoining of DNA strand breaks, rather than earlier repair processes like excision or polymerization.
• The mechanism of As inhibition likely involves the interaction of arsenite with vicinal dithiol groups, potentially inactivating DNA ligase III, which contains such groups.