A Missense Mutation in Rev7 Disrupts Formation of Polζ, Impairing Mouse Development and Repair of Genotoxic Agent-induced DNA Lesions

Maryam Khalaj,Abdolrahim Abbasi,Hiroshi Yamanishi,Kouyou Akiyama,Shuso Wakitani,Sotaro Kikuchi,Michiko Hirose,Misako Yuzuriha,Masaki Magari,Heba A Degheidy,Kuniya Abe,Atsuo Ogura,Hiroshi Hashimoto,Tetsuo Kunieda

doi:10.1074/jbc.m113.514752

Abstract

Repro22 is a mutant mouse produced via N-ethyl-N-nitrosourea-induced mutagenesis that shows sterility with germ cell depletion caused by defective proliferation of primordial germ cells, decreased body weight, and partial lethality during embryonic development. Using a positional cloning strategy, we identified a missense mutation in Rev7/Mad2l2 (Rev7(C70R)) and confirmed that the mutation is the cause of the defects in repro22 mice through transgenic rescue with normal Rev7. Rev7/Mad2l2 encodes a subunit of DNA polymerase ζ (Polζ), 1 of 10 translesion DNA synthesis polymerases known in mammals. The mutant REV7 did not interact with REV3, the catalytic subunit of Polζ. Rev7(C70R/C70R) cells showed decreased proliferation, increased apoptosis, and arrest in S phase with extensive γH2AX foci in nuclei that indicated accumulation of DNA damage after treatment with the genotoxic agent mitomycin C. The Rev7(C70R) mutation does not affect the mitotic spindle assembly checkpoint. These results demonstrated that Rev7 is essential in resolving the replication stalls caused by DNA damage during S phase. We concluded that Rev7 is required for primordial germ cell proliferation and embryonic viability and development through the translesion DNA synthesis activity of Polζ preserving DNA integrity during cell proliferation, which is required in highly proliferating embryonic cells.

Highlights

Rev7 encodes a subunit of Pol␨ for translesion DNA synthesis (TLS)
We concluded that Rev7 is required for primordial germ cell proliferation and embryonic viability and development through the translesion DNA synthesis activity of Pol␨ preserving DNA integrity during cell proliferation, which is required in highly proliferating embryonic cells
Our results show that Rev7 is required for primordial germ cells (PGCs) proliferation and embryonic viability and development through the TLS activity of Pol␨ to preserve DNA integrity during cell proliferation, which is required in highly proliferating embryonic cells

Summary

Background

Rev encodes a subunit of Pol␨ for translesion DNA synthesis (TLS). Results: We found a Rev mutation in mice that causes developmental defects and increases susceptibility for genotoxicity. The Rev7C70R mutation does not affect the mitotic spindle assembly checkpoint These results demonstrated that Rev is essential in resolving the replication stalls caused by DNA damage during S phase. DNA polymerase ␨ (Pol␨), is a member of the B family of DNA polymerases and consists of a catalytic and an accessory subunit encoded by Rev (official name, Rev3l) and Rev (official name, Mad2l2 for mitotic spindle assembly checkpoint protein MAD2B), respectively. A strong epistatic relationship occurs between Rev and Rev mutations, enhancing susceptibility to genotoxic agents These functions are conserved in vertebrates, studies using cultured cells with defects of these genes document greater complexity [11,12,13]. Our results show that Rev is required for PGC proliferation and embryonic viability and development through the TLS activity of Pol␨ to preserve DNA integrity during cell proliferation, which is required in highly proliferating embryonic cells

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION