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Smc5/6's multifaceted DNA binding capacities stabilize branched DNA structures.

ABSTRACT: Smc5/6 is an evolutionarily conserved SMC complex with roles in DNA replication and repair, as well as in viral DNA restriction. Understanding its multiple functions has been hampered by a lack of mechanistic studies on how the Smc5/6 complex associates with different types of DNA. Here we address this question by simultaneously visualizing the behavior of Smc5/6 on three types of DNA, namely double-stranded (ds) DNA, single-stranded (ss) DNA, and junction DNA formed by juxtaposed ss- and dsDNA, using correlative single-molecule fluorescence and force microscopy. We find that Smc5/6 displays distinct behaviors toward different types of DNA, dynamically associating with dsDNA while stably binding to junction DNA. Mechanistically, both the Nse1-3-4 subcomplex and ATP binding enhance the complex's dsDNA association. In contrast, Smc5/6's assembly onto ssDNA emanating from junction DNA, which occurs even in the presence high-affinity ssDNA binders, is aided by Nse1-3-4, but not by ATP. Moreover, we show that Smc5/6 protects junction DNA stability by preventing ssDNA annealing. The multifaceted DNA association behaviors of Smc5/6 provide a framework for understanding its diverse functions in genome maintenance and viral DNA restriction.

SUBMITTER: Chang JT

PROVIDER: S-EPMC9684126 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Smc5/6's multifaceted DNA binding capacities stabilize branched DNA structures.

Chang Jeremy T-H JT Li Shibai S Beckwitt Emily C EC Than Thane T Haluska Cory C Chandanani Joshua J O'Donnell Michael E ME Zhao Xiaolan X Liu Shixin S

Nature communications 20221123 1

Smc5/6 is an evolutionarily conserved SMC complex with roles in DNA replication and repair, as well as in viral DNA restriction. Understanding its multiple functions has been hampered by a lack of mechanistic studies on how the Smc5/6 complex associates with different types of DNA. Here we address this question by simultaneously visualizing the behavior of Smc5/6 on three types of DNA, namely double-stranded (ds) DNA, single-stranded (ss) DNA, and junction DNA formed by juxtaposed ss- and dsDNA, ...[more]

PMID: 36418314

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Project description:The structural and functional properties of G protein-coupled receptors (GPCRs) are often studied in a detergent micellar environment, but many GPCRs tend to denature or aggregate in short alkyl chain detergents. In our previous work [Lee, S., et al. (2016) J. Am. Chem. Soc. 138, 15425-15433], we showed that GPCRs in alkyl glucosides were highly dynamic, resulting in the penetration of detergent molecules between transmembrane α-helices, which is the initial step in receptor denaturation. Although this was not observed for GPCRs in dodecyl maltoside (DDM, also known as lauryl maltoside), even this detergent is not mild enough to preserve the integrity of many GPCRs during purification. Lauryl maltose neopentylglycol (LMNG) detergents have been found to have significant advantages for purifying GPCRs in a native state as they impart more stability to the receptor than DDM. To gain insights into how they stabilize GPCRs, we used atomistic molecular dynamics simulations of wild type adenosine A2A receptor (WT-A2AR), thermostabilized A2AR (tA2AR), and wild type β2-adrenoceptor (β2AR) in a variety of detergents (LMNG, DMNG, OGNG, and DDM). Analysis of molecular dynamics simulations of tA2AR in LMNG, DMNG, and OGNG showed that this series of detergents exhibited behavior very similar to that of an analogous series of detergents DDM, DM, and OG in our previous study. However, there was a striking difference upon comparison of the behavior of LMNG to that of DDM. LMNG showed considerably less motion than DDM, which resulted in the enhanced density of the aliphatic chains around the hydrophobic regions of the receptor and considerably more hydrogen bond formation between the head groups. This contributed to enhanced interaction energies between both detergent molecules and between the receptor and detergent, explaining the enhanced stability of GPCRs purified in this detergent. Branched detergents occlude between transmembrane helices and reduce their flexibility. Our results provide a rational foundation to develop detergent variants for stabilizing membrane proteins.

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Smc5/6's multifaceted DNA binding capacities stabilize branched DNA structures.

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Smc5/6's multifaceted DNA binding capacities stabilize branched DNA structures.

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