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

Thanh Thi Le, Johanna Ainsworth, Cristian Polo Rivera, Thomas Macartney, Karim P. M. Labib (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)
48 Downloads (Pure)


Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2LRR1 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 CUL2LRR1 . The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1. CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes. Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7. Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.
Original languageEnglish
Pages (from-to)2825-2842
Number of pages18
JournalBiochemical Journal
Issue number14
Early online date29 Jun 2021
Publication statusPublished - 23 Jul 2021


  • DNA replication
  • CMG helicase
  • CUL2LRR1
  • UBE2D
  • UBE2R
  • UBE2G
  • p97
  • ATPase

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry
  • Cell Biology


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