Journal: Nucleic Acids Research
Article Title: Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair
Figure Lengend Snippet: Acceleration of daughter strand unwinding and degradation by GATC sites flanking the mismatch. ( A ) Agarose gel analysis of nicking and unwinding of 0.5 nM circular DNA containing a single G/T mismatch at different positions and one or two GATC sites by 10 nM MutS, 10 nM MutL, 5 nM MutH, 5 nM UvrD, 200 nM Ssb and 0.1 units of ExoI. Early time points (2 and 4 min) showed the conversion of the closed circular DNA (lower band) to open-circular DNA (upper band) due to nicking by MutH. Later time points showed unwinding of nicked daughter strand by UvrD and degradation by ExoI starting from the 3′ end as indicated in the schematic drawings above the gel panels. ( B ) Quantification of the fraction of unnicked and nicked DNA for GT#1, GT#1b, GT#2 and GT#2b (mean ± SD, n = 3) with fit according to the unwinding model. Kinetic parameters obtained from the fit are tabulated in Supplementary Table S4. ( C ) Unwinding and excision of GT#1b pre-nicked with MutH alone (left panel), with MutH and Cas9 at site CrB such that nicks were on the same side of the mismatch (middle panel), and with MutH and Cas9 at site CrA such that the nicks flank the mismatch (right panel). ( D ) Quantification of unwinding (mean ± SD, n = 3) and fit with a function describing a single exponential increase. Kinetic parameters obtained from the fits are tabulated in Supplementary Table S4.
Article Snippet: ExoI and Ssb were purchased from New England Biolabs (Ipswich, USA) and Promega (Madison, USA), respectively.
Techniques: Agarose Gel Electrophoresis