Alkaline Bromodeoxyuridine (BrdU) Comet Assay to Detect Replication-Associated DNA Damage

Autor(es)

Ribeiro, Diego Luis ; Londero, James Eduardo Lago ; Martins, Davi Jardim ; Menck, Carlos Frederico Martins

• Tipo de documento: Artigo Científico
• Fonte: CURRENT PROTOCOLS; v. 5, n. 12, p. 26-pg., 2025-12-11.

Resumo

DNA replication is often challenged by endogenous and exogenous sources of DNA damage, which can stall replication forks and result in single-stranded DNA (ssDNA) gaps, double-strand breaks (DSBs), and genomic instability. Detecting DNA damage specifically in newly synthesized DNA strands is essential for understanding how eukaryotic cells respond to replication stress and continue the cell cycle progression through DNA repair or DNA damage tolerance (DDT) mechanisms. Here, we present an optimized and accessible protocol for the alkaline BrdU comet assay-a single-cell technique that combines bromodeoxyuridine (BrdU; a thymidine analog) pulse-labeling of newly synthesized DNA with the alkaline comet assay (single-cell gel electrophoresis) followed by fluorescence immunodetection. This method enables the specific detection and measurement of DNA strand breaks occurring in newly replicated DNA during and immediately after the S phase, even without cell synchronization, allowing researchers to differentiate replication-associated DNA damage from overall genomic damage. We provide detailed instructions for performing the assay using human cells (RPE-1 h-TERT TP53 KO) in vitro after exposure to DNA replication-stress-inducing agents, such as hydroxyurea (HU) and ultraviolet-C (UV-C) radiation. We also demonstrate its application in translesion-synthesis-deficient (Pol eta-deficient) human fibroblasts in vitro. Importantly, this protocol supports time-course chase experiments (e.g., 0, 1, 2, and 4 hr post-treatment) to monitor the kinetics of DNA damage in nascent DNA strands. This BrdU-based protocol offers high specificity, single-cell resolution, and cost-effectiveness, making it particularly valuable for laboratories studying replication stress, post-replication DNA repair proficiency, DDT, and/or genotoxic responses in unsynchronized human cells in vitro. This protocol also adheres to the Minimum Information for Reporting Comet Assay (MIRCA) guidelines and is aligned with the objectives of the International Comet Assay Working Group (ICAW), ensuring high reproducibility and standardization. (c) 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Alkaline BrdU comet assay to assess replication-associated DNA damage in unsynchronized human cells (RPE-1 h-TERT TP53 KO) in vitro Alternate Protocol 1: Alkaline BrdU comet assay to monitor replication-associated DNA damage dynamics in unsynchronized human cells (RPE-1 h-TERT TP53 KO) after HU and UV-C exposure and 0- to 2- hr chase Alternate Protocol 2: Alkaline BrdU comet assay to compare replication-associated DNA damage dynamics in translesion synthesis polymerase eta-deficient and complemented (XP-V comp) unsynchronized fibroblasts after UV-C exposure and 0- to 4-hr chase (AU)

• Processo FAPESP: 19/19435-3 - Papel de danos no DNA e função mitocondrial em envelhecimento vascular, imune e neurológico (DNA MoVINg)
• Beneficiário: Carlos Frederico Martins Menck
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 22/16390-1 - Estresse oxidativo e reparo de DNA em células humanas de Xeroderma Pigmentosum Variante expostas à radiação UVA
• Beneficiário: James Eduardo Lago Londero
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 20/02836-2 - Mecanismos de resistência de Glioblastoma ao antitumoral temozolomida em células cultivadas como esferoides multicelulares tumorais em três dimensões (3D) in vitro: o papel das polimerases de síntese translesão
• Beneficiário: Diego Luis Ribeiro
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 23/10452-8 - Mecanismos de resistência do Glioblastoma ao antitumoral Temozolomida em células cultivadas como modelos tridimensionais (3D) in vitro e in vivo: O papel das polimerases de Síntese de Translesão
• Beneficiário: Diego Luis Ribeiro
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Pós-Doutorado