Journal: Nucleic Acids Research
Article Title: Characterization of DNA synthesis catalyzed by bacteriophage T4 replication complexes reconstituted on synthetic circular substrates
Figure Lengend Snippet: Effect of an abasic site on DNA synthesis by reconstituted phage T4 replication complexes. ( A ) An abasic site blocks DNA synthesis by T4 DNA polymerase holoenzyme. 32 P-end-labeled 21mer #7 annealed with 120mer #3 (lanes 1, 3, 5, 7, 9, 11, 13 and 15) or with the same oligomer containing an abasic site at position 99 (120mer #4) (lanes 2, 4, 6, 8, 10, 12, 14 and 16) was incubated with either T4 gp43 (lanes 5, 6, 13 and 14) or the exonuclease-deficient gp43D219A (lanes 7, 8, 15 and 16) in the presence of gp44/62, gp45 and gp32 for the indicated times, and the products were separated in a 10% polyacrylamide gel + 6 M urea. Note that in the substrate with an abasic site, the first nucleotide to be incorporated into the 32 P-end-labeled 21mer is opposite the abasic site. ( B ) Block to lagging-strand synthesis imposed by the abasic site. Products of DNA synthesized by reconstituted T4 replication complexes in the presence of [α- 32 P]dCTP and 240mer circular ssDNA annealed with the 120mer ssDNA without (lanes 1 and 2) or with (lanes 3 and 4) abasic site were digested (lanes 2 and 4) or not (lanes 1 and 3) by Dra I and separated in a 6% polyacrylamide gel + 6 M urea along with marker DNA. Note that the 240mer circular ssDNA has a unique Dra I site. Upon Dra I cleavage of the replication products, the major 240mer fragment is formed along with other fragments that are located between the Dra I site and either the 3′ or 5′ ends of Okazaki fragments. Arrows show positions of the major 240-nt Dra I fragment and the 126-nt fragment located between the Dra I site and the 3′ end of the nascent fragment terminated by the abasic site. ( C ) DNA products were synthesized by reconstituted T4 replication complexes in the presence of [α- 32 P]dTTP and of 240mer circular ssDNA annealed with 120mer ssDNA without (lanes 1–3) or with (lanes 4–6) an abasic site, and separated in a 0.6% alkali gel.
Article Snippet: T4 DNA ligase, Dra I and polynucleotide kinase were from New England Biolabs.
Techniques: DNA Synthesis, Labeling, Incubation, Blocking Assay, Synthesized, Marker