The study introduces a method called single-cell 5-ethyl-2′-deoxyuridine sequencing (scEdU-seq) to quantify DNA replication speeds in single human cells. This method uses metabolic labeling with 5-ethyl-2′-deoxyuridine (EdU) and affinity capture of newly synthesized DNA fragments to detect nascent replicated DNA. The authors observed that DNA replication speed increases during the S phase of the cell cycle, with the highest speed occurring in late S phase. They found that transcription limits replication speed in early S phase, as transcription levels are highest in these regions. Inhibition of RNA polymerase II (RNAPII) transcription increased replication speed, suggesting that transcription-coupled DNA damage, which activates poly (ADP-ribose) polymerase (PARP), reduces replication speed. The study also showed that PARP inhibitors can reverse the decrease in replication speed caused by hyperactivation of PARP in cells lacking XRCC1, a protein required for efficient DNA damage repair. Overall, the findings highlight the complex interplay between DNA replication and transcription, with transcription-coupled DNA damage playing a crucial role in regulating replication speed.The study introduces a method called single-cell 5-ethyl-2′-deoxyuridine sequencing (scEdU-seq) to quantify DNA replication speeds in single human cells. This method uses metabolic labeling with 5-ethyl-2′-deoxyuridine (EdU) and affinity capture of newly synthesized DNA fragments to detect nascent replicated DNA. The authors observed that DNA replication speed increases during the S phase of the cell cycle, with the highest speed occurring in late S phase. They found that transcription limits replication speed in early S phase, as transcription levels are highest in these regions. Inhibition of RNA polymerase II (RNAPII) transcription increased replication speed, suggesting that transcription-coupled DNA damage, which activates poly (ADP-ribose) polymerase (PARP), reduces replication speed. The study also showed that PARP inhibitors can reverse the decrease in replication speed caused by hyperactivation of PARP in cells lacking XRCC1, a protein required for efficient DNA damage repair. Overall, the findings highlight the complex interplay between DNA replication and transcription, with transcription-coupled DNA damage playing a crucial role in regulating replication speed.