Enzymatic and DNA binding properties of purified WRN protein: high affinity binding to single-stranded DNA but not to DNA damage induced by 4NQO

Abstract

Mutations in the WRN gene result in Werner syndrome, an autosomal recessive disease in which many characteristics of aging are accelerated. A probable role in some aspect of DNA metabolism is suggested by the primary sequence of the WRN gene product. A recombinant His-tagged WRN protein (WRNp) was overproduced in insect cells using the baculovirus system and purified to near homogeneity by several chromatographic steps. This purification scheme removes both nuclease and topoisomerase contaminants that persist following a single Ni(2+)affinity chromatography step and allows for unambiguous interpretation of WRNp enzymatic activities on DNA substrates. Purified WRNp has DNA-dependent ATPase and helicase activities consistent with its homology to the RecQ subfamily of proteins. The protein also binds with higher affinity to single-stranded DNA than to double-stranded DNA. However, WRNp has no higher affinity for various types of DNA damage, including adducts formed during 4NQO treatment, than for undamaged DNA. Our results confirm that WRNp has a role in DNA metabolism, although this role does not appear to be the specific recognition of damage in DNA.

Inhibition of Werner Syndrome Helicase Activity by Benzo[c]phenanthrene Diol Epoxide dA Adducts in DNA Is Both Strand-and Stereoisomer-dependent

“…The turnover rate constants (k cat ) for initial rates of ATP hydrolysis measured in the plateau region (200 nM forked-duplex DNA) were 75 and 62 min Ϫ1 for 1-unadducted and 5-cis-S BcPh DNA effectors, respectively. These k cat values are in the same range as previously published values for WRN ATP hydrolysis using single-stranded DNA oligonucleotide effectors (43) under different reaction conditions. The similar K eff and k cat values for WRN ATP hydrolysis using BcPh DE-modified or unmodified forked-duplex molecules indicate that the presence of the BcPh DE adduct did not alter the allosteric effect of the bound DNA molecule on WRN ATP hydrolysis.…”

“…Proteins-Recombinant hexahistidine-tagged WRN protein (wildtype, WRN-E84A) was overexpressed using a baculovirus/Sf9 insect system and purified as described previously (43). UvrD (DNA helicase II) was overexpressed in Escherichia coli and purified as described previously (44).…”

The Exonucleolytic and Endonucleolytic Cleavage Activities of Human Exonuclease 1 Are Stimulated by an Interaction with the Carboxyl-terminal Region of the Werner Syndrome Protein

“…M. Gray (University of Washington, Seattle) and J. Campisi (Lawrence Berkeley National Laboratory, Berkeley, CA). Amplified WRN-encoding baculovirus was used to infect Sf9 cells for overexpression of WRN protein as described elsewhere (37). A recombinant hexahistidine-tagged carboxyl-terminal fragment of WRN (residues 940 -1432, designated C-WRN 940 -1432 ) was overexpressed in Escherichia coli and purified as described previously (38).…”

Structure of the human RECQ1 helicase reveals a putative strand-separation pin

“…RecQ helicases are ATP-and Mg 2ϩ -dependent enzymes that unwind DNA with a 3Ј to 5Ј polarity and, although with some differences, are capable of unwinding a variety of DNA structures other than standard B-form DNA duplexes such as forked duplexes, D-loops, triple helices, 3-or 4-way junctions, and G-quadruplex DNA (16)(17)(18)(19)(20)(21). Even when acting on the same substrates, they may do so by using different mechanisms (22).…”