Processing of G4 DNA by Dna2 Helicase/Nuclease and Replication Protein A (RPA) Provides Insights into the Mechanism of Dna2/RPA Substrate Recognition

Taro Masuda-Sasa,Piotr Polaczek,Xiao P Peng,Lu Chen,Judith L Campbell

doi:10.1074/jbc.m802244200

Abstract

The polyguanine-rich DNA sequences commonly found at telomeres and in rDNA arrays have been shown to assemble into structures known as G quadruplexes, or G4 DNA, stabilized by base-stacked G quartets, an arrangement of four hydrogen-bonded guanines. G4 DNA structures are resistant to the many helicases and nucleases that process intermediates arising in the course of DNA replication and repair. The lagging strand DNA replication protein, Dna2, has demonstrated a unique localization to telomeres and a role in de novo telomere biogenesis, prompting us to study the activities of Dna2 on G4 DNA-containing substrates. We find that yeast Dna2 binds with 25-fold higher affinity to G4 DNA formed from yeast telomere repeats than to single-stranded DNA of the same sequence. Human Dna2 also binds G4 DNAs. The helicase activities of both yeast and human Dna2 are effective in unwinding G4 DNAs. On the other hand, the nuclease activities of both yeast and human Dna2 are attenuated by the formation of G4 DNA, with the extent of inhibition depending on the topology of the G4 structure. This inhibition can be overcome by replication protein A. Replication protein A is known to stimulate the 5'- to 3'-nuclease activity of Dna2; however, we go on to show that this same protein inhibits the 3'- to 5'-exo/endonuclease activity of Dna2. These observations are discussed in terms of possible roles for Dna2 in resolving G4 secondary structures that arise during Okazaki fragment processing and telomere lengthening.

Highlights

Dna2 is that in Okazaki fragment processing (OFP)2 during DNA replication
Biochemical and genetic evidence suggests that Dna2 assists FEN1 in RNA/DNA primer removal during processing of a subset of Okazaki fragments with long 5Ј flaps and flaps with secondary structures arising because of excessive strand displacement by polymerase ␦ [5]
G-Quadruplex Structures—G-rich telomeric repeat sequences of eukaryotes from humans to yeast to protozoans are capable of interacting with monovalent cations to form both intramolecular and intermolecular G quadruplex structures in vitro. (The terminology used is as follows: G quadruplex, a fourstranded structure found in sequences rich in runs of guanines that is stabilized by formation and stacking of G quartets and can be formed from one, two, or four G-rich strands; G4 DNA, synonymous with G quadruplex; G quartet, square, planar arrangement of four guanines in a tetrad stabilized by Hoogsteen pairing and a monovalent cation.) We have used a yeast

Summary

Introduction

Dna2 is that in Okazaki fragment processing (OFP)2 during DNA replication. Biochemical and genetic evidence suggests that Dna2 assists FEN1 (flap endonuclease) in RNA/DNA primer removal during processing of a subset of Okazaki fragments with long 5Ј flaps and flaps with secondary structures arising because of excessive strand displacement by polymerase ␦ [5]. Gel shift assays indicated that scDna2 bound with high affin- bands, indicating the formation of multiple complexes containity to the yeast G4 DNA and bound G4 DNA preferentially over ing different molar ratios of protein to DNA (Fig. 4A, lanes the single-stranded DNA of the same sequence or a 5Ј flap 7–10).

Results

Conclusion