Purification of the yeast Slx5–Slx8 protein complex and characterization of its DNA-binding activity

SLX5 and SLX8 encode RING-finger proteins that were previously identified based on their requirement for viability in yeast cells lacking Sgs1 DNA helicase. Slx5 and Slx8 proteins are known to be required for genome stability and to physically interact in yeast extracts; however, their biochemical f...

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Main Authors:	Yang, Litao, Mullen, Janet R., Brill, Steven J.
Format:	Online
Language:	English
Published:	Oxford University Press 2006
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635298/

id	pubmed-1635298
recordtype	oai_dc
spelling	pubmed-16352982006-11-29 Purification of the yeast Slx5–Slx8 protein complex and characterization of its DNA-binding activity Yang, Litao Mullen, Janet R. Brill, Steven J. Nucleic Acid Enzymes SLX5 and SLX8 encode RING-finger proteins that were previously identified based on their requirement for viability in yeast cells lacking Sgs1 DNA helicase. Slx5 and Slx8 proteins are known to be required for genome stability and to physically interact in yeast extracts; however, their biochemical functions are unknown. To address this question we purified and characterized recombinant Slx5 and Slx8 proteins. Here we show that Slx5 and Slx8 form a heterodimeric complex with double-stranded DNA (dsDNA)-binding activity. Individually, only the Slx8 subunit displays this activity. Structure–function studies indicate that the DNA-binding activity requires only the N-terminal 160 amino acids of Slx8, but not its C-terminal RING-finger domain. Alleles of SLX8 that express the RING-finger domain alone show almost complete complementation in yeast indicating that this DNA-binding domain is not essential for this in vivo function. Consistent with these findings we show that Slx5 immunolocalizes to the nucleus and that a portion of the Slx8 protein co-fractionates with chromatin. These results suggest that Slx5–Slx8 may act directly on DNA to promote genome stability. Oxford University Press 2006-11 2006-10-04 /pmc/articles/PMC1635298/ /pubmed/17020915 http://dx.doi.org/10.1093/nar/gkl685 Text en © 2006 The Author(s)
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Yang, Litao Mullen, Janet R. Brill, Steven J.
spellingShingle	Yang, Litao Mullen, Janet R. Brill, Steven J. Purification of the yeast Slx5–Slx8 protein complex and characterization of its DNA-binding activity
author_facet	Yang, Litao Mullen, Janet R. Brill, Steven J.
author_sort	Yang, Litao
title	Purification of the yeast Slx5–Slx8 protein complex and characterization of its DNA-binding activity
title_short	Purification of the yeast Slx5–Slx8 protein complex and characterization of its DNA-binding activity
title_full	Purification of the yeast Slx5–Slx8 protein complex and characterization of its DNA-binding activity
title_fullStr	Purification of the yeast Slx5–Slx8 protein complex and characterization of its DNA-binding activity
title_full_unstemmed	Purification of the yeast Slx5–Slx8 protein complex and characterization of its DNA-binding activity
title_sort	purification of the yeast slx5–slx8 protein complex and characterization of its dna-binding activity
description	SLX5 and SLX8 encode RING-finger proteins that were previously identified based on their requirement for viability in yeast cells lacking Sgs1 DNA helicase. Slx5 and Slx8 proteins are known to be required for genome stability and to physically interact in yeast extracts; however, their biochemical functions are unknown. To address this question we purified and characterized recombinant Slx5 and Slx8 proteins. Here we show that Slx5 and Slx8 form a heterodimeric complex with double-stranded DNA (dsDNA)-binding activity. Individually, only the Slx8 subunit displays this activity. Structure–function studies indicate that the DNA-binding activity requires only the N-terminal 160 amino acids of Slx8, but not its C-terminal RING-finger domain. Alleles of SLX8 that express the RING-finger domain alone show almost complete complementation in yeast indicating that this DNA-binding domain is not essential for this in vivo function. Consistent with these findings we show that Slx5 immunolocalizes to the nucleus and that a portion of the Slx8 protein co-fractionates with chromatin. These results suggest that Slx5–Slx8 may act directly on DNA to promote genome stability.
publisher	Oxford University Press
publishDate	2006
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635298/
_version_	1611390494011555840

Purification of the yeast Slx5–Slx8 protein complex and characterization of its DNA-binding activity

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