Journal: Nucleic Acids Research

Article Title: Nucleotide excision repair of aflatoxin-induced DNA damage within the 3D human genome organization

doi: 10.1093/nar/gkae755

Figure Lengend Snippet: Experimental design and adaptation of tXR-seq for mapping nucleotide excision repair of AFB1–dG adducts. ( A ) Overview of the experimental design for in vivo excision assay, CPD/(6–4)PP XR-seq (4 h post-exposure) and AFB1–dG tXR-seq (4 h post-exposure). ( B ) Detection of excision products by excision assay after UVC (20 J/m 2 ) or AFB1 (40 μM) treatment in HepG2 cells. Excision products from one-quarter of a 150 mm petri dish of UVC-treated HepG2 cells (0.5 h post-exposure) were loaded into the gel, while for AFB1-treated HepG2 cells (4 h post-exposure), excision products from two 150 mm petri dishes were used in the excision assay. M, DNA size marker; C, non-damaged control. ( C ) Sequencing library construction workflow for CPD/(6–4)PP XR-seq and AFB1–dG tXR-seq. Red asterisk indicates DNA bulky adduct. Excision products, released during nucleotide excision repair, are precipitated with TFIIH/XPG antibodies, extracted and ligated to adaptors. Then the adaptor-containing oligomers are precipitated with damage IP. Photolyases are used for the reversal of UV damage for CPD/(6–4)PP XR-seq. For AFB1–dG tXR-seq, Dpo4 (pink) is the sulfolobus DNA polymerase IV, a Y-family DNA polymerase known for its ability to bypass various DNA lesions. It is used for bypassing the AFB1–dG damage during the primer extension before the PCR amplification. ( D ) Optimization of AFB1–dG tXR-seq library preparation. Lanes show results using different anti-AFB1 antibodies (6A10 and AFA-1) and translesion synthesis DNA polymerases (Dpo4 and polymerase ζ). Lanes 1, 2, and 7 are non-damaged controls. Libraries were analyzed by 10% native polyacrylamide gel electrophoresis. Red star marks PCR products containing inserts; black arrow indicates adapter dimers.

Article Snippet: Sulfolobus DNA polymerase IV (Dpo4) (Cat. No. M0327) and DNA polymerase ζ (Cat. No. 51) were purchased from NEB and Enzymax, respectively.

Techniques: In Vivo, Excision Assay, Marker, Control, Sequencing, Amplification, Translesion Synthesis, Polyacrylamide Gel Electrophoresis

Journal: Genes and Environment

Article Title: Analysis of nucleotide insertion opposite urea and translesion synthesis across urea by DNA polymerases

doi: 10.1186/s41021-022-00236-3

Figure Lengend Snippet: DNA synthesis and selective nucleotide incorporation opposite urea (Ua) by DNA polymerase ζ (panel A), DNA polymerase η (panel B) and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) (panel C). DNA polymerase ζ (1.7 ng), DNA polymerase η (0.4 ng) and Dpo4 (0.4 ng) ware incubated with templates containing G (lanes 1–5) or Ua (lanes 6–10) and 100 μM of each of the four dNTPs (lanes 5 and 10) or 100 μM of a single dNTP (N = C, G, A, or T) (lanes 1–4 and 6–9). Lane M contained no enzyme and are negative controls

Article Snippet: Dpo4 was purchased from Trevigen (Gaithersburg, USA).

Techniques: DNA Synthesis, Incubation