The Rtf1-RTS1 fork-stalling system

The Rtf1-RTS1 fork-stalling system has been established in vivo and provides an opportunity to understand the link between the replication fork blockage and chromosomal rearrangements (Figure 1-10A) [19]. RTS1 sequences are a form of programmed RFBs that have evolved for specific purposes. In nature, programmed RFBs are generally direction-dependent and facilitate a polar arrest of fork coming from one direction and allow unblocked passage of forks coming from the opposite direction [28, 57-58]. The Rtf1-RTS1 fork-stalling system is based on a ~850bp replication termination sequence (RTS1) that is required as a polar replication fork barrier near the mat locus and blocks fork coming from the centromere side during S.

pombe mating type switching [56]. Genetic screens identified that RTS1 sequences associate with several proteins to pause the replication fork progression; Four genes have been shown to interact with the RTS1 sequences, swi1, swi3, rtf1, and rtf2 [59, 116]. Swi1 and swi3 are components of the replisome and activate the S-phase checkpoint pathway for the stabilisation of stalled forks. Rtf1, a Myb-like DNA binding protein and Rtf2, a PCNA interacting protein are transcription termination factors that interfere with the replicative helicase. Two distinct cell-types (h⁺ or h^-) express distinct sexual markers in fission yeast. Mating type switching from one sexual type to another occurs near the mating type locus, mat1. An imprint at the mat1 locus of S. pombe initiates the replication-coupled recombination mechanism required for the mating-type switching. The formation of the imprint involves a lagging-strand-induced replication pause at mat1 and depends on unidirectional fork progression ensured by the RTS system. This imprint is maintained until the next cell cycle when the leading-strand replication complex is arrested at the imprint. The resolution of the stalled fork in the leading-strand requires HR-based process which

allows the recombination between mat1 and one of the silent donor cassettes mat2P or mat3M, leading to the mating-type switching [56].

We have used the natural properties of RTS1 sequences as a barrier that blocks replication fork movement in one direction for the development of the Rtf1-RTS1 fork-stalling system. As described above at programmed RFBs, a polar fork arrest is caused by Rtf1, a non-histone protein, binding to the RTS1 sequence. In our laboratory, a series of fork-arrest-induced assays have been established, here, our discussion focuses mainly on the inverted repeat (RuraR) and the palindrome (RuiuR) system [49, 50]. In the inverted repeat RuraR assay, an ura4 marker is introduced flanked by two inverted RTS1 sequences (Figure 1-10B). Palindromes are a type of inverted repeat sequences separated by a few base pairs. Palindrome RuiuR assay contains two ura4⁺ marker genes in inverted orientation with a 14bp DNA sequence spacer between them and flanked by RTS1 inverted repeats (Figure 1-10B). Both constructs have been established separately on chromosome III of S. pombe and was confirmed to generate acentric and dicentric chromosomes by chromosomal rearrangements. By controlling the expression of the rtf1⁺ gene, the progressing forks can be artificially inhibited by Rtf1 binding to the RTS1 sequences. To regulate the expression of rtf1⁺ genes, an inducible thiamine-repressible promoter nmt41 was introduced to replace the rtf1⁺ gene promoter. The expression of Rtf1 protein is detected after 12 hours of thiamine removal and reaches a peak at around 16 hours.

Fork arrest within either RuraR or RuiuR was predicted. Indeed, when rtf1⁺ was expressed, more than 94% of the forks stopped at the outer part of each RTS1 barriers of RuraR or RuiuR assays and the recovery of the arrested forks were mediated by a DSB-independent mechanism and involved the recruitment of repair proteins at the RTS1 site (see details below). Subsequent fork-arrest based rearrangement events

occurred at high frequencies (∼15–25%), allowing further molecular analysis. This Rtf1-RTS1 fork-stalling system also provided a direct evidence that fork failure leads to genome rearrangements.

Figure 1-10. (A) Schematics of the Rtf1-RTS1 fork-stalling system. A replication fork is stalled initially by the RTS1 sequence bound by the inducible Rtf1 protein, which leads to fork collapse.

Restart of the fork can lead to chromosomal rearrangements and generate acentric and dicentric chromosomes. RTS1 sequence is shown as the blue square; white triangle indicates the orientation for replication fork stalling. (B) Inverted repeat (RuraR) and palindrome (RuiuR) assay. We used the Rtf1-RTS1 fork-stalling system to initiate recombination events. RuraR and RuiuR assays are based on development of the Rtf1-RTS1 fork-stalling system established on ChrIII in Schizosaccharomyces pombe. RuraR assay is one single ura4 gene flanked by RTS1 inverted

repeats, while RuiuR assay contains two ura4 genes in inverted orientation with 14bp DNA sequence spacer flanked by RTS1 inverted repeats. Blue squares RTS1 sequence; white triangle indicates the orientation for stalling a replication fork and the orientation of ura4 gene. (C) Spot test to check cell viability. Cells suffer viability loss and miss-segregation due to RuraR and RuiuR construct established on the essential chromosome III of S. pombe. Cells containing RuraR and

RuiuR assay on ChrIII lost viability after replication fork arrest (“pause on” growth). Cells that

underwent miss-segregation in mitosis showed the presence of lagging chromosomes visualised by DAPI and Calcofluor stain.

1.4.4 Observations on chromosomal rearrangements from our previous model on