Unwinding of a DNA Triple Helix by the Werner and Bloom Syndrome Helicases

Robert M Brosh,Alokes Majumdar,Shital Desai,Ian D Hickson,Vilhelm A Bohr,Michael M Seidman

doi:10.1074/jbc.m006784200

Abstract

Bloom syndrome and Werner syndrome are genome instability disorders, which result from mutations in two different genes encoding helicases. Both enzymes are members of the RecQ family of helicases, have a 3' --> 5' polarity, and require a 3' single strand tail. In addition to their activity in unwinding duplex substrates, recent studies show that the two enzymes are able to unwind G2 and G4 tetraplexes, prompting speculation that failure to resolve these structures in Bloom syndrome and Werner syndrome cells may contribute to genome instability. The triple helix is another alternate DNA structure that can be formed by sequences that are widely distributed throughout the human genome. Here we show that purified Bloom and Werner helicases can unwind a DNA triple helix. The reactions are dependent on nucleoside triphosphate hydrolysis and require a free 3' tail attached to the third strand. The two enzymes unwound triplexes without requirement for a duplex extension that would form a fork at the junction of the tail and the triplex. In contrast, a duplex formed by the third strand and a complement to the triplex region was a poor substrate for both enzymes. However, the same duplex was readily unwound when a noncomplementary 5' tail was added to form a forked structure. It seems likely that structural features of the triplex mimic those of a fork and thus support efficient unwinding by the two helicases.

Highlights

Despite the obvious importance of genetic stability, the mammalian genome has an abundance of sequences that are potentially destabilizing
In this report we describe the activity of the BLM and WRN helicases on a pyrimidine-purine:pyrimidine triple helix and corresponding duplex structures
The BLM and WRN enzymes are DNA-stimulated ATPases with 3Ј–5Ј-helicase activity. They are active against short duplexes on the M13 single strand substrate, unwind longer duplex substrates in the presence of replication protein A (RPA) [20], [38], and are potently inhibited by minor groove binding compounds such as netropsin and distamycin [69]

Summary

Introduction

Despite the obvious importance of genetic stability, the mammalian genome has an abundance of sequences that are potentially destabilizing. The mutant gene in Bloom syndrome patients encodes a RecQ family helicase with 3Ј–5Ј polarity it lacks an exonuclease activity (34 – 36) It is found associated with other proteins involved in DNA metabolism [37], including RPA [38]. Whereas the defining function of these and all other helicases is the unwinding of duplex nucleic acid, recent work indicates that the BLM and WRN enzymes are active on alternate DNA structures. They can unwind tetrahelical structures that can form in stretches of G-rich DNA (G DNA) [40, 41]. The possibility that the genomic instability of the WS and BS disorders might be due to a failure to resolve alternate DNA structures has generated considerable interest [47]

Methods

Results

Conclusion