Reconstitution of human CMG helicase ubiquitylation by CUL2LRR1 and multiple E2 enzymes

Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2(LRR1) is required to ubiquitylate the MCM7 subunit of the CMG helicase. Here, we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase. Using purified human proteins, including a panel of E2 ubiquitin-conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2(LRR1). The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting sz subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1. CUL2(LRR1) firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes. Subsequently, CUL2(LRR1) activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7. Thereby, CUL2(LRR1) converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.

Automated summary

Cullin ubiquitin ligases drive CMG-MCM7 ubiquitylation in human cells, converting the helicase into a substrate for p97 unfoldase.