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R-loop proteins present a stable and robust blockade to the progression of a DNA replication fork during S-phase. The consequences of this block can include mutagenesis and other irreversible chromosomal catastrophes, causing genomic instability and disease. As such, further investigation into the molecular mechanisms underlying R-loop protein resolution is warranted. The critical role of non-replicative accessory helicases in R-loop protein resolution has increasingly come into light in recent years. Such helicases include the Pif1-family, monomeric helicases that have been studied in many different contexts and that have been ascribed to a multitude of separable protective functions in the cell. In this chapter, we present protocols to study R-loop protein resolution by Pif1 helicase at stalled replication forks using purified proteins, both at the biochemical and single-molecule level. Our system uses recombinant proteins expressed in Saccharomyces cerevisiae but could apply to prac ....
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