Poly dt

IDCC (N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide), 6-IATR (6-iodoacetamidotetramethylrhodamine) and 5-IATR (5-iodoacetamidotetramethylrhodamine) were gifts from J. Corrie (NIMR, London, U.K.) [19 –21 ]. dT₃₅, dT_70, poly(dT) and other chemicals were from Sigma-Aldrich unless otherwise stated. poly(dT) has an average length ~500 bases and in all measurements, its concentration was calculated in terms of 70-base units, approximating to the SSB binding sites. AddAB helicases (wild type from Bacillus subtilis and helicase-inactive mutant, AddA^HB(A-K36A)) were gifts from Mark Dillingham (University of Bristol, U.K.) [22 (link)].
Biotin-TEG-DNA, linear dsDNA of different lengths but without Chi-sequences, for the AddAB assay, was prepared by PCR from the pCERoriD 4907 bp plasmid [6 (link)] and amplifying the sequences using 5’-biotinylated forward primer (5’bio-TEG-GGG TAT TGT TTG TTC CCT GAG CGC G 3’) and various reverse primers (585 bp: 5’-GTC TGC CTC CAC CGC GGC CAC G 3’, 985 bp: 5’- CTG TGC CAT GAC GGA GGA TGA TG 3’, 1216 bp: 5’-AGG AAA ACC GGT ACA GAA CTG CA 3’, and 1458 bp: 5’-CGG CAT ACC TCA GTG GCG TGG AG 3’). Bio-DNAs were gel purified using Qiagen Gel Extraction kit.

Poly(dT), poly(dA), ATP, and Tween-20 were obtained from Sigma-Aldrich (St. Louis, MO, USA). HsRad51 was purified according to a previously described procedure [49 (link)]. Poly(dεA) was synthesized by chemical modification of poly(dA) using chloroacetaldehyde (Aldrich), according to a previous method [50 (link)]. The degree of modification was approximately 93%, determined by spectroscopy using the formula provided by Ledneva et al. [51 (link)]. Concentrations were determined based on UV absorption, utilizing the following extinction coefficients: ε_263nm = 8520 M⁻¹ cm⁻¹ for Poly(dT), ε_257nm = 3800 M⁻¹ cm⁻¹ for poly(dεA), and ε_260nm = 15,400 M⁻¹ cm⁻¹ for ATP.