Fig. 2: HMCES suppresses DSB formation during NEIL3-dependent ICL repair.

a, pICL^AP was replicated with [α−³²P]dATP in mock- or HMCES-depleted extracts supplemented with rHMCES^WT or rHMCES^C2A, as indicated. Replication intermediates were separated on a native agarose gel and visualized by autoradiography. SC, supercoiled; OC, open circular; WP, well products. Red arrowheads indicate linear species; blue arrowheads indicate well-products.

b, Experimental strategy to synchronize AP-ICL unhooking by NEIL3-depletion and add back. p97i is added to replication reactions to prevent CMG unloading and accumulate replication forks that have converged at the ICL. rNEIL3 is added to stalled forks to activate unhooking.

c, pICL^AP was replicated with [α−³²P]dATP as described in (b) using the NEIL3- or NEIL3- and HMCES-depleted extracts shown in Extended Data Fig. 2a. Replication intermediates were analyzed as in a.

d, pICL^AP was replicated in mock- or HMCES-depleted egg extracts (shown in Extended Data Fig. 2g) supplemented with rHMCES, as indicated. Replication reactions were separated by SDS-PAGE and blotted for phospho-CHK1 and MCM6 (loading control).