Characterization of Werner syndrome protein DNA helicase activity: Directionality, substrate dependence and stimulation by replication protein A

Abstract

Werner syndrome is an inherited disease characterized by premature aging, genetic instability and a high incidence of cancer. The wild type Werner syndrome protein (WRN) has been demonstrated to exhibit DNA helicase activity in vitro. Here we report further biochemical characterization of the WRN helicase. The enzyme unwinds double-stranded DNA, translocating 3'-->5' on the enzyme-bound strand. Hydrolysis of dATP or ATP, and to a lesser extent hydrolysis of dCTP or CTP, supports WRN-catalyzed strand-displacement. K m values for ATP and dATP are 51 and 119 microM, respectively, and 2.1 and 3.9 mM for CTP and dCTP, respectively. Strand-displacement activity of WRN is stimulated by single-stranded DNA-binding proteins (SSBs). Among the SSBs from Escherichia coli, bacteriophage T4 and human, stimulation by human SSB (human replication protein A, hRPA) is the most extensive and occurs with a stoichiometry which suggests direct interaction with WRN. A deficit in the interaction of WRN with hRPA may be associated with deletion mutations that occur at elevated frequency in Werner syndrome cells.

WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repair

“…WRN exonuclease activity is suppressed by interaction with p53 (Brosh et al ., 2001) or BLM (von , and stimulated by interaction with Ku70/80 (Li & Comai, 2001) or phosphorylation . WRN helicase activity is stimulated by interaction with p53 (Yang et al, 2002), replication protein A (RPA) (Shen et al, 1998), telomere repeat binding factor 2 (TRF2) and phosphorylation . Given that WRN can reside in a large multiprotein complex , it is likely that its enzymatic activities are regulated in vivo by phosphorylation and by interactions with other proteins.…”

Asymmetry of DNA replication fork progression in Werner's syndrome

“…Our results provide strong experimental support for a role for WRN in unwinding double helical structures in the DNA template via its 3 ′− 5 ′ helicase activity (Gray et al ., 1997;Suzuki et al ., 1997;Shen et al ., 1998;Kamath Loeb et al ., 2001), or perhaps promoting reverse branch migration of the junction away from a stalled fork. Although WRN is a member of the RecQ family of helicases (Chakraverty & Hickson, 1999;Yu et al ., 1996;Shen & Loeb, 2000) including BLM, none of these other helicases appears able to compensate fully for lack of WRN activity.…”

Werner's Syndrome Protein Is Required for Correct Recovery after Replication Arrest and DNA Damage Induced in S-Phase of Cell Cycle

“…Studies from yeast show that DNA replication does not proceed normally in absence of RecQ helicase function (Stewart et al, 1997) and in Xenopus laevis the ortholog of WRN is absolutely required for proper formation of replication foci (Yan et al, 1998). Functional interaction between proteins involved in DNA replication, such as replication protein A (RPA) and DNA polymerase ␦, and WRN also have been reported (Shen et al, 1998;Brosh et al, 1999;Kamath-Loeb et al, 2000). Furthermore, in yeast, the RecQlike proteins seem involved in suppression of hyperrecombination, S-phase checkpoint control, and correct DNA segregation (Gangloff et al, 1994, Watt et al, 1995Stewart et al, 1997;Davey et al, 1998;Yamagata et al, 1998;Frei and Gasser, 2000).…”