Resolution of Holliday Junction Recombination Intermediates by Wild-type and Mutant IntDOT Proteins

Overview

Journal J Bacteriol

Publisher American Society for Microbiology

Specialty Microbiology

Date 2011 Jan 11

PMID 21216992

Citations 4

Authors

Seyeun Kim

Jeffrey F Gardner

Affiliations

Soon will be listed here.

Abstract

CTnDOT encodes an integrase that is a member of the tyrosine recombinase family. The recombination reaction proceeds by sequential sets of genetic exchanges between the attDOT site in CTnDOT and an attB site in the chromosome. The exchanges are separated by 7 base pairs in each site. Unlike most tyrosine recombinases, IntDOT exchanges sites that contain different DNA sequences between the exchange sites to generate Holliday junctions (HJs) that contain mismatched bases. We demonstrate that IntDOT resolves synthetic HJs in vitro. Holliday junctions that contain identical sequences between the exchange sites are resolved into both substrates and products, while HJs that contain mismatches are resolved only to substrates. This result implies that resolution of HJs to products requires the formation of a higher-order nucleoprotein complex with natural sites containing IntDOT. We also found that proteins with substitutions of residues (V95, K94, and K96) in a putative alpha helix at the junction of the N and CB domains (coupler region) were defective in resolving HJs. Mutational analysis of charged residues in the coupler and the N terminus of the protein did not provide evidence for a charge interaction between the regions of the protein. V95 may participate in a hydrophobic interaction with another region of IntDOT.

Citing Articles

Resolution of Mismatched Overlap Holliday Junction Intermediates by the Tyrosine Recombinase IntDOT.

Ringwald K, Yoneji S, Gardner J J Bacteriol. 2017; 199(10).

PMID: 28242723 PMC: 5405215. DOI: 10.1128/JB.00873-16.

The N-terminus of IntDOT forms hydrophobic interactions during Holliday Junction resolution.

Kolakowski A, Gardner J Plasmid. 2016; 87-88:10-16.

PMID: 27422335 PMC: 5121019. DOI: 10.1016/j.plasmid.2016.07.003.

The Integration and Excision of CTnDOT.

Wood M, Gardner J Microbiol Spectr. 2015; 3(2):MDNA3-0020-2014.

PMID: 26104696 PMC: 4480416. DOI: 10.1128/microbiolspec.MDNA3-0020-2014.

Roles of Exc protein and DNA homology in the CTnDOT excision reaction.

Keeton C, Gardner J J Bacteriol. 2012; 194(13):3368-76.

PMID: 22505687 PMC: 3434738. DOI: 10.1128/JB.00359-12.

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