Crystallization and preliminary X-ray studies of I-PpoI: A nuclear, intron-encoded homing endonuclease from Physarum polycephalum

✍ Scribed by Karen E. Flick; Diane Mchugh; Barry L. Stoddard; Joe Don Heath; Kathryn M. Stephens; Raymond J. Monnat Jr.

• Book ID: 105356502
• Publisher: Cold Spring Harbor Laboratory Press
• Year: 2008
• Tongue: English
• Weight: 449 KB
• Volume: 6
• Category: Article
• ISSN: 0961-8368
• DOI: 10.1002/pro.5560061226

✦ Synopsis

Abstract

The homing endonuclease I‐__Ppo__I is encoded by an optional third intron, Pp LSU 3, found in nuclear, extrachromosomal copies of the Physarum polycephalum 26s rRNA gene. This endonuclease promotes the lateral transfer or “homing” of its encoding intron by recognizing and cleaving a partially symmetric, 15 bp homing site in 26s rDNA alleles that lack the Pp LSU 3 intron. The open reading frame encoding I‐__Ppo__I has been subcloned, and the endonuclease has been overproduced in E. coli. Purified recombi‐nant I‐__Ppo__I has been co‐crystallized with a 21 bp homing site DNA duplex. The crystals belong to space group P3~1~21, with unit cell dimensions a = b = 114 Å, c = 89 Å. The results of initial X‐ray diffraction experiments indicate that the asymmetric unit contains an enzyme homodimer and one duplex DNA molecule, and that the unit cell has a specific volume of 3.4 Å^3^/dalton. These experiments also provide strong evidence that I‐__Ppo__I contains several bound zinc ions as part of its structure.