Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks

Hyun-Suk Kim,Jac A Nickoloff,Yuehan Wu,Elizabeth A Williamson,Gurjit Singh Sidhu,Brian L Reinert,Aruna S Jaiswal,Gayathri Srinivasan,Bhavita Patel,Kimi Kong,Sandeep Burma,Suk-Hee Lee,Robert A Hromas

doi:10.1074/jbc.m116.758235

Abstract

Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5′ end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5′-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5′ end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5′-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5′ end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks.

Highlights

The replicating cell perpetually suffers from DNA damage from both endogenous and exogenous sources
Because EEPD1 is a 5Ј-endonuclease that is recruited to stressed replication forks and interacts with Exo1 [31], we hypothesized that EEPD1 could cleave replication fork structures to generate the needed free 5Ј-DNA ends upon which Exo1 can act [11, 12, 16, 24, 27, 28]
Mre11 probably has no other role in 5Ј end resection, as its major activity is a 3Ј- to 5Ј-exonuclease, the opposite of what is required for 5Ј end resection [16, 28]

Summary

Edited by Patrick Sung

Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5؅ end resection, mediated by exonuclease complexes, one of which contains Exo. We show that the 5؅-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands This cleavage creates the structure that Exo requires for 5؅ end resection and HR initiation. Unlike a DNA double-strand break, a stalled replication fork lacks a free DNA end from which one of the exonuclease complexes can initiate end resection (1–3, 14 –16). As EEPD1 has N-terminal RvuA-like domains, an uncharacterized nuclease domain in the DNase I superfamily, and interacts with Exo1 [31], we hypothesized that EEPD1 could cleave replication fork structures to provide the free 5Ј end to promote Exo1-mediated 5Ј end resection [11, 28, 31]. We report here that EEPD1 can cleave replication fork structures to permit Exo1-mediated 5Ј end resection and that both EEPD1 and Exo are required for proper replication fork restart after stalling

Results

Discussion

Experimental Procedures