Figure 3.

RFWD3 is essential for DNA synthesis across different polymerase-stalling DNA lesions

(A) pCTRL^ssDNA or pCPD^ssDNA were incubated in non-licensing egg extracts in the presence of [α-³²P]dATP. Samples were analyzed on a denaturing polyacrylamide gel following AatII-ApoI digest. Top scheme: extension products generated by AatII-ApoI digest.

(B) pCPD^ssDNA was incubated in mock-, Polη-, REV1-, or RFWD3-depleted non-licensing extracts. Samples were analyzed as in (A). Quantification of the relative intensity of the −1 product for three independent experiments is shown in the lower panel. Intensity of the −1 product was quantified for each lane and normalized to the maximum value. Error bars represent SEM.

(C) pICL^pt was replicated in mock- or RFWD3-depleted extracts (with either the RFWD3-N or the RFWD3-F antibody), in the presence of pQuant. Reaction samples were analyzed by native agarose gel electrophoresis.

(D) Intermediates generated by AflIII digest on pICL^pt.

(E) Samples from (C) were digested with AflIII and analyzed on a denaturing polyacrylamide gel.