Topoisomerase IIIα and Bloom’s helicase can resolve a mobile double Holliday junction substrate through convergent branch migration

doi:10.1073/pnas.0604873103

Topoisomerase IIIα and Bloom’s helicase can resolve a mobile double Holliday junction substrate through convergent branch migration

Department of Biochemistry, Duke University Medical Center, Durham, NC 27710

Communicated by James C. Wang, Harvard University, Cambridge, MA, June 10, 2006 (received for review April 18, 2006)

Abstract

It has long been suspected that a double Holliday junction (dHJ) could be resolved by a topoisomerase partnered with a helicase by convergent branch migration of the HJs. Genetic analysis of yeast TOP3 and SGS1 has lent considerable evidence to the notion that the protein products of these genes are involved in just such a process, although biochemical analysis of the metabolism of a dHJ has been hindered by the lack of a substrate that adequately replicates the endogenous structure. We have synthesized a dHJ substrate that recapitulates many of the features of an endogenous dHJ and represents a much earlier intermediate in the resolution pathway. Here, we show that Drosophila topoisomerase IIIα (Topo IIIα) and Blm (a homolog of Sgs1) are capable of resolving this substrate to non-cross-over products and that this activity is stimulated by replication protein A (RPA). We investigated the ability of other Drosophila topoisomerases to perform this reaction in concert with Blm and RPA and discovered that this resolution activity is unique to Topo IIIα. Examination of the mechanism of resolution reveals that Topo IIIα, Blm, and RPA resolve this substrate by convergent migration of the two HJs toward each other, collapsing the dHJ. This mechanism stands in contrast to classic resolvase activities that use a structure-specific endonuclease to cleave the HJs.

Footnotes

*To whom correspondence should be addressed at:
Department of Biochemistry, Duke University Medical Center, DUMC Box 3711, Durham, NC 27710.
E-mail: hsieh{at}biochem.duke.edu
Author contributions: J.L.P. and T.-s.H. designed research; J.L.P. performed research; J.L.P. and J.W. contributed new reagents/analytic tools; J.L.P. and T.-s.H. analyzed data; and J.L.P. and T.-s.H. wrote the paper.
Conflict of interest statement: No conflicts declared.
Abbreviations:

Abbreviations:

Topo III,

topoisomerase III;

dHJ,

double Holliday junction;

DHJS,

Double Holliday Junction Substrate;

RPA,

replication protein A;

CO,

cross-over;

NCO,

non-CO;

hBLM,

human BLM;

hRPA,

human RPA;

hTopo,

human Topo;

T7 endo I,

T7 endonuclease I.