Fig. 1: Inhibition of DNA licensing by geminin in an in vitro assay.
From: Geminin inhibits DNA replication licensing by sterically blocking CDT1-MCM2 interactions
a Simplified cartoon of human DNA licensing. ATP binding and hydrolysis highlighted in orange. The addition of ORC6 in our reactions means that the ORC6-dependent pathway is expected to be dominant. Coloured bars identify complexes associated with each pre-RC assay wash type. b Schematic of pre-RC assay protocol. c Pre-RC assay with reactions assembled ± geminin (GemFL). GemFL (*green) stabilises CDC6 (*red) and inhibits CDT1ΔN (*blue) and ORC6 (*purple) interaction within LS elutions. With HS elutions, GemFL inhibits MCM2-7 loading. d Quantification of MCM2-7 loading in the HS-reactions of (c, lanes 3 and 4). Data normalised to the mean pre-RC reaction and analysed by two-tailed paired t-test (**p = 0.0042). Mean of three biological repeats ±SD. e In solution EM analysis of DH formation. 500–1000 micrographs were collected for each condition, and particles picked automatically. Two rounds of 2D classification were used to select for DHs (representative classes shown) and used to determine DH/micrograph. Mean of two independent reactions. f Anti-CDT1 western blot of LS pre-RC reactions ± GemFL. g Quantification of CDT1 signal in (f). Data expressed as a percentage of the CDT1 input, normalised and compared to the control pre-RC mean and analysed by two-tailed paired t-test, **p = 0.0052. Mean ± SD of three biological repeats. h Exclusion of CDT1ΔN from pre-RC reactions inhibits MCM2-7 loading (*red = CDC6, *blue = CDT1ΔN and *purple = ORC6). Representative of three biological repeats. i Domain organisation of our standard CDT1ΔN construct (residues 158–546), the CDT1 middle helical (CDT1MHD, residues 158–396) and C-terminal helical (CDT1CHD, residues 391–546) domains. j Pre-RC assay of the CDT1 MHD and CHD at 1× and 5× concentration (compared to CDT1ΔN control). k Quantification of CDC6 signal in (j). Reactions compared to the −CDT1ΔN control using one-way ANOVA with mixed-effects analysis and Dunnett’s multiple comparisons test. ****p < 0.0001 and *p = 0.0158. Mean ± SD. l Quantification of HS MCM2-7 loading in (j). Reactions compared to the -CDT1ΔN control using one-way ANOVA with mixed-effects analysis and Dunnett’s multiple comparisons test. ****p < 0.0001 and *p = 0.0319. Mean ± SD of three biological repeats. Source data are provided as a Source Data file.
