Proteasome-dependent degradation of replisome components regulates faithful DNA replication.

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF(Pof3) (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.

Coordinated degradation of replisome components ensures genome stability upon replication stress in the absence of the replication fork protection complex.

The stabilization of the replisome complex is essential in order to achieve highly processive DNA replication and preserve genomic integrity. Conversely, it would also be advantageous for the cell to abrogate replisome functions to prevent inappropriate replication when fork progression is adversely perturbed. However, such mechanisms remain elusive. Here we report that replicative DNA polymerases and helicases, the major components of the replisome, are degraded in concert in the absence of Swi1, a subunit of the replication fork protection complex. In sharp contrast, ORC and PCNA, which are also required for DNA replication, were stably maintained. We demonstrate that this degradation of DNA polymerases and helicases is dependent on the ubiquitin-proteasome system, in which the SCF(Pof3) ubiquitin ligase is involved. Consistently, we show that Pof3 interacts with DNA polymerase ε. Remarkably, forced accumulation of replisome components leads to abnormal DNA replication and mitotic catastrophes in the absence of Swi1. Swi1 is known to prevent fork collapse at natural replication block sites throughout the genome. Therefore, our results suggest that the cell elicits a program to degrade replisomes upon replication stress in the absence of Swi1. We also suggest that this program prevents inappropriate duplication of the genome, which in turn contributes to the preservation of genomic integrity.

Lsd1 and lsd2 control programmed replication fork pauses and imprinting in fission yeast.

In the fission yeast Schizosaccharomyces pombe, a chromosomal imprinting event controls the asymmetric pattern of mating-type switching. The orientation of DNA replication at the mating-type locus is instrumental in this process. However, the factors leading to imprinting are not fully identified and the mechanism is poorly understood. Here, we show that the replication fork pause at the mat1 locus (MPS1), essential for imprint formation, depends on the lysine-specific demethylase Lsd1. We demonstrate that either Lsd1 or Lsd2 amine oxidase activity is required for these processes, working upstream of the imprinting factors Swi1 and Swi3 (homologs of mammalian Timeless and Tipin, respectively). We also show that the Lsd1/2 complex controls the replication fork terminators, within the rDNA repeats. These findings reveal a role for the Lsd1/2 demethylases in controlling polar replication fork progression, imprint formation, and subsequent asymmetric cell divisions.

Mus81 is essential for sister chromatid recombination at broken replication forks.

Recombination is essential for the recovery of stalled/collapsed replication forks and therefore for the maintenance of genomic stability. The situation becomes critical when the replication fork collides with an unrepaired single-strand break and converts it into a one-ended double-strand break. We show in fission yeast that a unique broken replication fork requires the homologous recombination (HR) enzymes for cell viability. Two structure-specific heterodimeric endonucleases participate in two different resolution pathways. Mus81/Eme1 is essential when the sister chromatid is used for repair; conversely, Swi9/Swi10 is essential when an ectopic sequence is used for repair. Consequently, the utilization of these two HR modes of resolution mainly relies on the ratio of unique and repeated sequences present in various eukaryotic genomes. We also provide molecular evidence for sister recombination intermediates. These findings demonstrate that Mus81/Eme1 is the dedicated endonuclease that resolves sister chromatid recombination intermediates during the repair of broken replication forks.