Measuring Low-Picomolar Apparent Binding Affinities by Minigel Electrophoretic Mobility Shift

Lewis, Karen A.; Altschuler, Sarah E.; Wuttke, Deborah S.

doi:10.1007/978-1-4939-8793-1_29

Cited by 1 publication

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References 10 publications

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“…We purified a recombinant N-terminal fragment of ESCO1 fused to GFP (ESCO1 1-200). Holding a 32 P-radiolabeled single-stranded 60 base primer at a constant concentration, we titrated in the ESCO1 fusion (26, 27). Consistent with direct binding of ESCO1 to the DNA, the fusion protein resulted in greatly reduced mobility of the DNA fragment (Fig.…”

Section: Resultsmentioning

confidence: 99%

The intrinsically disordered tail of ESCO1 binds DNA in a charge-dependent manner

Schoen,

Chen,

Rankin

2023

Preprint

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ESCO1 is an acetyltransferase enzyme that regulates chromosome organization and gene expression. It does this by modifying the Smc3 subunit of the Cohesin complex. Although ESCO1 is enriched at the base of chromatin loops in a Cohesin-dependent manner, precisely how it interacts with chromatin is unknown. Here we show that the basic and intrinsically disordered tail of ESCO1 binds DNA with very high affinity, likely through electrostatic interaction. We show that neutralization of positive residues in the N-tail reduces both DNA binding in vitro and association of the enzyme with chromatin in cells. Additionally, disruption of the chromatin state and charge distribution reduces chromatin bound ESCO1. Strikingly, defects in DNA binding do not affect total SMC3 acetylation or sister chromatid cohesion, suggesting that ESCO1-dependent acetylation can occur independently of direct chromatin association. We conclude that the intrinsically disordered tail of ESCO1 binds DNA with both high affinity and turnover, but surprisingly, ESCO1 catalytic activity occurs independently of direct DNA binding by the enzyme.

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Section: Resultsmentioning

confidence: 99%