Analysis of chromatin unwinding by the UvrD DNA helicase

In an effort to understand how nuclear processes occur in the context of repressive chromatin structures found in vivo, we have investigated the ability of the E. coli UvrD helicase to unwind nucleosomal substrates in vitro. UvrD is a DNA repair helicase whose eukaryotic functional counterpart is defective in the skin cancer disorder, Xeroderma Pigmentosum. Model chromatin templates containing the 5S nucleosome positioning sequence were generated that include linear mononucleosomes and subsaturated nucleosomal arrays. UvrD was incubated with naked and nucleosomal DNA templates in the presence of MgATP, and then ssDNA products were separated from dsDNA substrates using native PAGE followed by detection and quantitation via Southern Blotting and phosphorimaging. Our results demonstrate that UvrD can unwind mononucleosomal DNA to similar extents (100%) as the corresponding naked DNA template. UvrD also unwound nucleosomal arrays that were 70% saturated, to reduced extents (50%). Above this nucleosome loading level, unwinding was significantly reduced, indicating a threshold level for nucleosome‐imposed steric inhibition of helicase‐promoted unwinding. Histone acetylation had no effect on extents of unwinding of chromatin substrates. We are currently trying to image UvrD in the process of unwinding chromatin using atomic force microscopy. These studies will help establish how helicases contend with chromatin structure and what mechanisms the cell uses to modify chromatin structure so that DNA‐related enzymes can gain access to them. The authors gratefully acknowledge funding support provided by Midwestern University.