Controlling tetramer formation, subunit rotation and DNA ligation during Hin-catalyzed DNA inversion.

Yong Chang,Reid C Johnson

doi:10.1093/nar/gkv565

Abstract

Two critical steps controlling serine recombinase activity are the remodeling of dimers into the chemically active synaptic tetramer and the regulation of subunit rotation during DNA exchange. We identify a set of hydrophobic residues within the oligomerization helix that controls these steps by the Hin DNA invertase. Phe105 and Met109 insert into hydrophobic pockets within the catalytic domain of the same subunit to stabilize the inactive dimer conformation. These rotate out of the catalytic domain in the dimer and into the subunit rotation interface of the tetramer. About half of residue 105 and 109 substitutions gain the ability to generate stable synaptic tetramers and/or promote DNA chemistry without activation by the Fis/enhancer element. Phe106 replaces Phe105 in the catalytic domain pocket to stabilize the tetramer conformation. Significantly, many of the residue 105 and 109 substitutions support subunit rotation but impair ligation, implying a defect in rotational pausing at the tetrameric conformer poised for ligation. We propose that a ratchet-like surface involving Phe105, Met109 and Leu112 within the rotation interface functions to gate the subunit rotation reaction. Hydrophobic residues are present in analogous positions in other serine recombinases and likely perform similar functions.

Highlights

Tight control over any reaction involving severing of DNA strands is of critical importance because of the risk of permanent chromosome damage
The present study provides new insights into intrinsic controls over both the formation of the chemically active tetramer and the subunit rotation reaction using the Hin serine recombinase system as the model
In addition we have examined the effects of substitutions at Met109, whose side chain is predicted to insert into a shallower pocket on the surface of the catalytic domain in the Hin dimer (Figure 2A)

Summary

Introduction

Tight control over any reaction involving severing of DNA strands is of critical importance because of the risk of permanent chromosome damage. This is true of DNA recombination reactions mediated by the serine recombinase family of enzymes [1,2]. DNA molecules, and the cleaved DNA ends are held together by a noncovalent hydrophobic interface between recombinase polypeptides. This interface is dynamic, enabling the subunits with their covalently attached DNA ends to rotate about each other to generate the recombinant DNA configuration. The present study provides new insights into intrinsic controls over both the formation of the chemically active tetramer and the subunit rotation reaction using the Hin serine recombinase system as the model

Methods

Results

Conclusion