The first set of three substrates consisted of M viral DNA to which oligonucleotides were annealed.A, DNA helicase assay using a nucleotide oligomer annealed to M viral DNA as a substrate.The three oligonucleotides used were either completely complementary or contained a nucleotide or noncomplementary region, in addition to the complementary nucleotides.Oligonucleotides were labeled with P at their end before annealing to the viral DNA.Products were separated by electrophoresis through a nondenaturing polyacrylamide gel and visualized by autoradiography.B, DNA helicase assay using a bp linear dsDNA as substrate.Reactions were carried out in the absence or presence of ATP or in the presence of an ATP regeneration system, as indicated.They were analyzed as described in A, and the same control reactions were included.The positions of the substrate and the radiolabeled reaction product are indicated to the left of the autoradiogram.C, DNA helicase assay using a branched dsDNA as substrate.Its substrate is a dsDNA end.Detection was with alkaline phosphatasecoupled goat antirabbit antibodies.The percentage of surviving cells as measured by their colonyforming ability is plotted as a function of the gray dose.C, clonogenic survival assay of the same cell lines shown in B after treatment with increasing concentrations of mitomycin C.Many superfamily members have DNA helicase activity.Possibly, the seven conserved amino acid sequence motifs that define the superfamily provide a general activity, of which helicase activity could be a subset. This more general activity could be the ability to translocate along DNA at the expense of ATP hydrolysis. Providing processivity to joint formation might be especially important in the context of chromatin.Biol. Chem. doi. jbc. Access the most updated version of this article at http:www.jbc.orgcontent When a correction for this article is posted Click here to choose from all of JBCs email alerts This article cites references, of which can be accessed free at http:www.jbc.orgcontent.full.htmlreflist Downloadedfromhttp: www.jbc.orgbyguestonSeptember, These nuclease activities are likely to be important for recombination, repair, and genomic stability.Many of these genes, including RAD, RAD, RAD, RAD, RAD, RAD, RAD, RDH, MRE, and XRS, show epistasis and are collectively known as the RAD epistasis group.Mutants of the RAD group also have defects of varying degrees in mitotic and meiotic recombination, which are initiated via DNA doublestranded break formation.Because meiotic recombination is essential for the proper segregation of homologous chromosomal pairs during meiosis I, the RAD group mutants often exhibit severe meiotic abnormalities, including inviability. Extensive genetic evidence in yeast indicates that DNA doublestranded breaks are processed exonucleolytically, yielding overhanging singlestranded. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with U.The RAD group genes may be divided into two categories.The first class consists of the RAD, MRE, and XRS genes, whose protein products are thought to be involved in the nucleolytic processing of DNA doublestranded breaks. The second category of the RAD group genes includes RAD, RAD, RAD, RAD, RAD, and RDH, whose products nucleate onto the ssDNA tails generated from break processing and then mediate the formation of heteroduplex DNA between the recombining chromosomes, also has a role in heteroduplex DNA formation remains to be established.