Structure and Function of a Novel ATPase that Interacts with Holliday Junction Resolvase Hjc and Promotes Branch Migration

Abstract

Holliday junction (HJ) is a hallmark intermediate in DNA recombination and must be processed by dissolution (for double HJ) or resolution to ensure genome stability. Although HJ resolvases have been identified in all domains of life, there is a long-standing effort to search in prokaryotes and eukarya for proteins promoting HJ migration. Here, we report the structural and functional characterization of a novel ATPase, Sulfolobus islandicus PilT N-terminal-domain-containing ATPase (SisPINA), encoded by the gene adjacent to the resolvase Hjc coding gene. PINA is conserved in archaea and vital for S. islandicus viability. Purified SisPINA forms hexameric rings in the crystalline state and in solution, similar to the HJ migration helicase RuvB in Gram-negative bacteria. Structural analysis suggests that ATP binding and hydrolysis cause conformational changes in SisPINA to drive branch migration. Further studies reveal that SisPINA interacts with SisHjc and coordinates HJ migration and cleavage.

The archaeal ATPase PINA interacts with the helicase Hjm via its carboxyl terminal KH domain remodeling and processing replication fork and Holliday junction

“…We solved a novel structure of SisPINA revealing that the last 70 residues form a classical type II KH domain. Based on the results reported here and published previously by our group ( 39 ), we propose a model illustrating the mechanisms and roles of PINA in stalled replication fork repair through recombinational repair pathway.…”

“…The fragment containing pyrEF-lacs was utilized to transform S. islandicus E233S cells by electroporation as previously described ( 41 ). The screening procedure for the strain with chromosomally-coded N-terminal 6 × His-tagged SisPINA (Sis/pMID-SisPINA-in-situ-N-His-T) was the same as for Sis/pMID-SisPINA-T ( 39 ).…”

“…Vectors for SisPINA and SisHjc expression were constructed as previously described in Zhai et al . ( 39 ). The vector for SisHjm expression was constructed as described in Song et al .…”

“…Recently, we have identified and characterized a novel ATPase from Sulfolobus islandicus , designated SisPINA (PIN domain-containing ATPase from S. islandicus ) ( 39 ). The protein is highly conserved among archaeal species; every archaeal strain that has been sequenced possesses a homolog of SisPINA, an indication of its essential role in Archaea.…”

“…We showed that SisPINA is able to drive HJ migration in vitro . The protein interacts physically and functionally with the HJ-specific endonuclease (resolvase), Hjc, and coordinates the HJ processing ( 39 ). Structural analysis suggested that ATP binding and hydrolysis cause conformational change of SisPINA which promotes HJ branch migration.…”