Dual Control of Yen1 Nuclease Activity and Cellular Localization by Cdk and Cdc14 Prevents Genome Instability

摘要

class="head no_bottom_margin" id="sec1title">IntroductionBefore every cell division, our genetic material needs to be duplicated and assembled into two equal packages that can be faithfully transmitted to the next generation. The temporal coordination of molecular events such as DNA replication, repair, and chromosome segregation is therefore critical for the maintenance of genome integrity. Homologous recombination (HR), although essential for the repair of DNA breaks and the restoration of damaged replication forks, can occasionally create a block to chromosome segregation by generating covalently linked DNA intermediates such as Holliday junctions (HJs) (). These joint molecules (JMs) constitute a physical connection between sister chromatids or homologous chromosomes, and their timely removal is needed to avoid any interference with normal chromosome segregation.Cells possess a battery of helicases and nucleases that can deal with HR intermediates. For example, in S. cerevisiae there are two major pathways for the processing of late HR intermediates. The first, mediated by the Sgs1-Top3-Rmi1 (STR) complex, specializes in the elimination of double HJs (dHJs) that arise from HR-mediated strand break repair (). For this, the RecQ-family helicase Sgs1 catalyzes the convergent branch migration of two HJs, resulting in a hemicatenate structure that is decatenated by the type I topoisomerase Top3. A related mechanism of double HJ “dissolution” occurs in human cells, driven by BLM-TopoIIIα-RMI1-RMI2 (BTR complex) and is important for the avoidance of sister chromatid exchanges and loss of heterozygosity (). Indeed, STR/BTR play an important role in limiting crossover (CO) formation by directing the products of recombination to noncrossovers (NCOs). The second pathway for the processing of late HR intermediates involves two structure-selective endonucleases: in yeast, these are the XPF-family endonuclease Mus81-Mms4 () and the XPG-family member Yen1 (). Again, there is a good analogy with human systems, where MUS81-EME1 and GEN1 are important for promoting Holliday junction “resolution” (). These reactions, unlike STR/BTR, have the potential to promote both the formation of NCO and CO products. Therefore, the choice of pathway for the removal of late HR intermediates directs the outcome of repair, with NCO pathways being the preferred choice in mitotic cells ().How the processing of recombination intermediates is biased toward NCOs in mitosis has been the focus of extensive research. In sgs1Δ mutants, the elimination of JMs is delayed until G2/M, becomes dependent on Mus81-Mms4, and results in increased CO formation (). Consistent with these observations, sgs1Δ mus81Δ double mutants are synthetically lethal due to the accumulation of toxic HR intermediates (), indicating that Mus81-Mms4 resolves HR intermediates that escape the actions of the STR complex at earlier stages of the cell cycle. The DNA repair deficiency of mus81Δ and mms4Δ mutants is exacerbated by further deletion of YEN1, and mus81Δ yen1Δ double mutants display both aberrant chromosome segregation and reduced mitotic CO formation (). Thus, Yen1 provides an additional mechanism for JM resolution.The apparent hierarchy among the STR complex, Mus81-Mms4, and Yen1 for DNA damage repair is supported by observations showing that Mus81-Mms4 and Yen1 are sequentially activated in two waves that occur late in the cell cycle. Cdk/Cdc5-mediated phosphorylation of Mms4 drives the hyperactivation of Mus81-Mms4 at the G2/M transition, whereas S phase phosphorylation of Yen1 holds this protein in an inactive state until it is activated at anaphase (). Intuitively, such a regulatory mechanism creates a window during S/G2 in which the NCO-promoting pathways have preferential access to the majority of repair intermediates. Any persistent dHJs, and single HJs that might arise from replication fork processing, would then be captured by Mus81-Mms4 in G2/M or by Yen1 in anaphase, thus ensuring the segregation of DNA. The temporal restriction of Mus81-Mms4 and Yen1 activities to late stages of the cell cycle may also be important to protect against the unscheduled cleavage of DNA replication intermediates that arise during S phase.Although the biochemical properties of Mus81-Mms4 have been studied extensively, the biochemical properties of Yen1 (759 amino acids) or its human ortholog GEN1 (908 amino acids) have yet to be determined. Indeed, the analysis of Yen1 has been restricted to immunoprecipitates, whereas most studies of GEN1 were carried out with a purified N-terminal fragment (GEN1^1-527) of the protein (). Like other members of the XPG family of 5′-flap endonucleases, Yen1/GEN1 exhibit 5′-flap and fork cleavage activities, but, unique to this nuclease family, Yen1 and GEN1 cleave HJs by the introduction of symmetrically related nicks across the junction to produce ligatable nicked duplex products.Yen1 is a target of the S phase cyclin-dependent kinase Cdk, and its subcellular localization is cell-cycle-regulated in a Cdk-dependent manner (). However, we know very little about the mechanism of Yen1 inhibition during S phase, how it is activated at anaphase, or how these dynamic cycles of inactivation/activation are coupled with cell-cycle progression. Here, we investigate the mechanism of Yen1 regulation, in terms of its biochemical activation and subcellular localization, and show how both are linked to cell-cycle control. Importantly, we demonstrate that Cdk and the mitotic exit phosphatase Cdc14 () constitute the “off” and “on” switches for Yen1 function, respectively. Employing Yen1 mutants that are refractory to Cdk-dependent inhibition and are constitutively active throughout the cell cycle, we show that premature activation of Yen1 results in DNA damage sensitivity and increased loss of heterozygosity. The purification of full-length Yen1, in both the phosphorylated and nonphosphorylated states, revealed that the mechanistic basis of inhibition lies in modulation of the DNA binding activity of the protein. As such, Yen1 is uniquely regulated at two distinct levels, by both biochemical inhibition and by nuclear exclusion, until anaphase when the protein is activated and undergoes nuclear entry in response to Cdc14-mediated dephosphorylation.