EEPD1: Breaking and Rescuing the Replication Fork

The faithful duplication of an entire genome is a complex affair requiring the coordinated action of the DNA replisome to unwind and synthesize DNA at replication forks. Unfortunately, exposure to chemicals or radiation can damage DNA strands, and this damage can stall DNA replication forks, resulting in genome instability, tumorigenesis, or cell death. To rescue stalled replication forks, cells have evolved a comprehensive network of DNA repair pathways that allow them to recover from these impediments (Fig 1) [1,2]. For example, either the direct chemical reversal of a damaged DNA base or DNA translesion synthesis allows a stalled replication fork to resume replication without disassembling the replisome. Alternatively, replication fork regression can generate a chicken foot structure, which provides the stalled strand with an undamaged sister chromatid to use as a template for DNA synthesis. This allows the replisome to bypass the damaged site. Fork regression also provides the cell an opportunity to repair DNA damage before converting chicken foot structures back to replication forks. However, because chicken foot structures resemble Holliday junctions (HJ), prolonged or unregulated fork regression may lead to DNA cleavage by HJ nucleases and DNA double-stranded breaks (DSBs). Collapsed replication forks are frequently associated with DSB formation. Even though DSBs can be repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ), DSBs are often the source of increased genomic instability [3,4]. For this reason, the recent discovery by Hromas, Nickoloff, and collaborators of a novel nuclease involved in DSB formation at stalled replication forks is quite interesting [5]. This team initiated their search for enzymes that are important for DNA damage repair by screening for genes induced by the topoisomerase IIα poison VP-16. They found that one of the up-regulated genes encodes a previously uncharacterized human protein named Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1). The absence of EEPD1 slowed the rate of replication fork progression after hydroxyurea (HU) treatment, indicating that EEPD1 is important for replication fork recovery from replication stress. In addition, HR frequency was significantly reduced in EEPD1-deficient cells, suggesting that EEPD1 may have a role in repairing stalled replication forks by HR. If this is the case, one would predict that the decrease in HR efficiency would lead to the accumulation of unrepaired DSBs. Surprisingly, just the opposite occurred: DSBs decreased in EEPD1 deficient cells, as indicated by reduced γH2AX focus formation and the amount of tail moment detected by an alkaline comet assay. Therefore, EEPD1 is required for generating DSBs in response to replication stress. A regressed replication fork containing an HJ-like chicken foot structure could be nicked on two opposite strands by an HJ resolvase to create a DSB [1,2]. Even though EEPD1 contains a DNA binding domain similar to that found in the bacterial HJ binding protein RuvA, in vitro EEPD1 does not cleave HJ like an HJ