Holliday junction resolution: regulation in space and time

Abstract

Holliday junctions (HJs) can be formed between sister chromatids or homologous chromosomes during the recombinational repair of DNA lesions. A variety of pathways act upon HJs to remove them from DNA, in events that are critical for appropriate chromosome segregation. Despite the identification and characterization of multiple enzymes involved in HJ processing, the cellular mechanisms that regulate and implement pathway usage have only just started to be delineated. A conserved network of core cell-cycle kinases and phosphatases modulate HJ metabolism by exerting spatial and temporal control over the activities of two structure-selective nucleases: yeast Mus81-Mms4 (human MUS81-EME1) and Yen1 (human GEN1). These regulatory cycles operate to establish the sequential activation of HJ processing enzymes, implementing a hierarchy in pathway usage that ensure the elimination of chromosomal interactions which would otherwise interfere with chromosome segregation. Mus81-Mms4/EME1 and Yen1/GEN1 emerge to define a special class of enzymes, evolved to satisfy the cellular need of safeguarding the completion of DNA repair when on the verge of chromosome segregation.

Keywords: Cdc5; Cell-cycle; DNA repair; EME1; GEN1; Mms4; Mus81; Nuclease; PLK1; Recombination; Resolvase; Slx1; Slx4; Yen1.

Fig. 1

DNA centric view showing three distinct pathways of Holliday junction processing. The BTR complex disengages dHJs, using the mechanism of “dissolution”, to generate NCO recombinants. MUS81-EME1 and SLX1-SLX4 interact to form the SLX-MUS complex which resolves both single and double HJs by endonucleolytic cleavage to generate COs and NCOs. GEN1 provides a separate pathway of HJ resolution.

Fig. 2

Cell-cycle centric view of HJ processing. During DNA repair, single or double HJs (sHJ or dHJ) are formed between sister chromatids or homologous chromosomes (for convenience, the diagram depicts two homologs). The STR (yeast) or BTR (human) complexes operate early during the cell cycle to dissolve dHJs and generate non-crossover recombinants. If DNA damage occurs late during the cell cycle or if HJs escape the attention of STR/BTR and persist until S/G2 phase, resolution enzymes ensure HJ processing and safeguard chromosome segregation.

Fig. 3

Spatio-temporal regulation of HJ processing enzymes. In S. cerevisiae, cell cycle kinases Cdk and Cdc5 transiently phosphorylate and activate Mus81-Mms4 at the G2/M transition. Cdk1-mediated phosphorylation of Yen1 prevents its nuclear accumulation and biochemical activation during S-phase and early stages of mitosis. Cyclin degradation and Cdc14-mediated dephosphorylation activate Yen1 during anaphase. In human cells, a similar regulatory network uses a different molecular mechanism to prevent the actions of MUS81-EME1 and GEN1 during S-phase and early G2. At the G2/M transition, M-phase CDK activity promotes the interaction of MUS81-EME1 with SLX1-SLX4 leading to the formation of an active SLX-MUS HJ resolvase. GEN1, which is excluded from the nucleus during early stages of the cell cycle, gains access to chromatin upon CDK1-mediated nuclear envelope breakdown.